Tripeptide prodrug compounds

ABSTRACT

The prodrug of the invention is a modified form of a therapeutic agent and comprises a therapeutic agent, an oligopeptide of three amino acids, a stabilizing group and, optionally, a linker group. The prodrug is cleavable by a trouase enzyme such as Thimet oligopeptidase. Also disclosed are methods of making and using the prodrug compounds.

INTRODUCTION

1. Technical Field

The present invention is directed to new compounds useful as prodrugs,and methods for making them. Such prodrugs may be used for treatment ofdisease, especially tumors, in patients.

2. Background

Many therapeutic agents, such as anthracyclines and vinca alkaloids, areespecially effective for the treatment of cancers. However, thesemolecules are often characterized in vivo by an acute toxicity,especially a bone marrow and mucosal toxicity, as well as a chroniccardiac toxicity in the case of the anthracyclines and chronicneurological toxicity in the case of the vinca alkaloids. Similarly,methotrexate may be used for the treatment of inflammatory reactions,such as rheumatic diseases, but its high toxicity limits itsapplications. Development of more and safer specific antitumor agents isdesirable for greater effectiveness against tumor cells and a decreasein the number and severity of the side effects of these products(toxicity, destruction of non-tumor cells, etc.). Development of morespecific anti-inflammatory agents is also desirable.

In order to minimize toxicity problems, therapeutic agents areadvantageously presented to patients in the form of prodrugs. Prodrugsare molecules capable of being converted to drugs (active therapeuticcompounds) in vivo by certain chemical or enzymatic modifications oftheir structure. For purposes of reducing toxicity, this conversionshould be confined to the site of action or target tissue rather thanthe circulatory system or non-target tissue. Prodrugs are oftencharacterized by a low stability in blood and serum, however. This isdue to the presence of enzymes in blood and serum that degrade, andconsequently may activate, the prodrugs before the prodrugs can reachthe desired sites within the patient's body.

A desirable class of prodrugs that overcomes such problems has beendisclosed in Patent Cooperation Treaty International Publication No. WO96/05863 and in U.S. Pat. No. 5,962,216, both incorporated herein byreference. Further useful prodrug compounds and methods of making suchprodrugs are desirable, however, as are methods of making the prodrugs.

Prodrugs that display a high specificity of action, a reduced toxicity,and an improved stability in blood especially relative to prodrugs ofsimilar structure (especially the closest structure) that have existedin the public domain are particularly desirable.

SUMMARY OF THE INVENTION

The compound of the invention is a prodrug form of a therapeutic agent,in which the therapeutic agent is linked directly or indirectly to anoligopeptide of three amino acids, which in turn, is linked to astabilizing group. The prodrugs of the invention display a highspecificity of action, a reduced toxicity, an improved stability inserum and blood, and move into target cells minimally unless activatedby a target cell associated enzyme. Additionally, the compounds arepreferably cleaved by a trouase, such as TOP, at a rate of cleavage thatis a fractional portion of the rate of cleavage ofSuc-βAla-Leu-Ala-Leu-Dox.

The present invention also relates to a pharmaceutical compositioncomprising the compound according to the invention and optionally apharmaceutically acceptable carrier, adjuvant, vehicle or the like.Articles of manufacture comprising the prodrugs of the invention arealso described. Thus, the invention includes a diagnosis or assay kitemploying a compound of the invention.

Further methods of designing a prodrug and of decreasing toxicity andimproving safety index by modifying a therapeutic agent to create aprodrug are disclosed. Such modification provides an improvedtherapeutic index of the prodrug as compared to the free therapeuticagent. Several methods of making prodrugs of the invention are alsodescribed.

The present invention further includes methods of treating a medicalcondition by administering the prodrug of the invention to a patient ina therapeutically effective amount.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1D are a table of abbreviations, names, and structures.

FIG. 2 is an exemplary scheme of cleavage of a prodrug of the inventionin the extracellular vicinity of the target cell and within the targetcell.

FIG. 3 illustrates a solid phase synthesis of Fmoc-Met-Ala-Leu, atypical intermediate of the invention.

FIG. 4 illustrates a solution phase “Fmoc-route” synthesis ofMethyl-succinyl-Met-Ala-Leu, a typical intermediate of the invention.

FIG. 5 illustrates an “Fmoc route” synthesis of a salt form ofSuc-Met-Ala-Leu-DOX, a typical compound of the invention.

FIG. 6 illustrates an “Ester route” synthesis of a salt form ofSuc-Met-Ala-Leu-DOX, a typical compound of the invention.

FIG. 7 illustrates a synthesis of an amino-protected Met-Ala-Leu-DOX, atypical intermediate of the invention.

FIG. 8 illustrates an “Allyl ester route” synthesis of a salt form ofSuc-Met-Ala-Leu-DOX, a typical compound of the invention.

FIG. 9 illustrates a “Resin route” synthesis of Suc-Met-Ala-Leu-DOX, atypical compound of the invention.

FIG. 10 is a graph of the plasma levels of Suc-Leu-Ala-Leu-Dox and itsmetabolites at 1 and 4 hours after administration of a singleintravenous bolus dose of the prodrug.

FIG. 11 is a graph of the amount of Suc-Leu-Ala-Leu-Dox and itsmetabolites present in the urine collected 0–2 and 2–24 hours after theadministration of a single intravenous bolus of the prodrug.

FIG. 12 is a graph of the plasma levels of Suc-Met-Ala-Leu-Dox and itsmetabolites at 1 and 4 hours after administration of a singleintravenous bolus dose of the prodrug.

FIG. 13 is a graph of the amount of Suc-Met-Ala-Leu-Dox and itsmetabolites present in the urine collected 0–2 and 2–24 hours after theadministration of a single intravenous bolus of the prodrug.

FIG. 14 is a graph of the Percent Body Weight Change of either micetreated with Suc-Leu-Ala-Leu-Dox or mice receiving the vehicle control.

FIG. 15 is a graph illustrating the increase in Mean Days of Survival(MDS) in mice treated with Suc-Leu-Ala-Leu-Dox as compared with micegiven the vehicle control.

FIG. 16 is a graph of the rate of tumor growth in LS174T xenograftedmice either treated with Suc-Leu-Ala-Leu-Dox or given the vehiclecontrol.

FIG. 17 illustrates the removal of free therapeutic agent through theuse of scavenging resin or beads.

DETAILED DESCRIPTION

Abbreviations

-   ACN=Acetonitrile-   Aib=Aminoisobutyric acid-   All=Allyl-   Aloc=Allyloxycarbonyl-   Amb=4-(Aminomethyl)benzoic acid-   APP=3-Amino-3-phenylpropionic acid-   DCC=N,N′-Dicyclohexylcarbodiimide-   Boc=t-butyloxycarbonyl-   Cap=amino caproic acid-   DBN=1,5 Diazabicyclo[4.3.0]non-5-ene-   DBO=1,4 Diazabicyclo[2.2.2]octane-   DBU=1,8-Diazabicyclo[5.4.0]undec-7-ene-   DCM=Dichloromethane-   DIC=N,N′-Diisopropylcarbodiimide-   DIEA=Diisopropylethylamine-   Dg=Diglycolic Acid-   DMF=Dimethylformamide-   Dnr=Daunorubicin-   Dox=Doxorubicin-   Et₂O=diethyl ether-   Fmoc=9-Fluorenylmethyloxycarbonyl-   Gl=Glutaric Acid-   HATU=O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate-   HBTU=2-(1H-Benzotriazole-1-yl)1,1,3,3-tetramethyluronium-hexafluorophosphate-   HEPES=Hydroxethylpiperidine-   HOBt=N-Hydroxybenzotriazole-   HPLC=High pressure liquid chromatography-   MeOH=Methanol-   MeOSuc=Methyl hemisuccinate or methyl hemisuccinyl-   MTD=Maximum tolerated dose-   NAA=3-Amino-4,4-diphenylbutyric Acid-   Nal=2-Naphthylalanine-   Naph=1,8-Naphthalene dicarboxylic acid-   Nle=Norleucine-   NMP=N-methylpyrrolidine-   Nva=Norvaline-   PAM resin=4-hydroxymethylphenylacetamidomethyl-   PEG=Polyethylene glycol-   Pyg=Pyroglutamic acid-   Pyr=3-Pyridylalanine-   RD=repeat dose-   RD-MTD=repeat dose maximum tolerated dose-   RT, rt=Room temperature-   SD=single dose-   SD-MTD=single dose maximum tolerated dose-   Suc=Succinyl Acid/Succinyl-   TCE=trichloroethyl-   TFA=trifluoroacetic acid-   THF=Tetrahydrofuran-   Thi=2-Thienylalanine-   Thz=Thiazolidine-4-carboxylic acid-   Tic=Tetrahydroisoquinoline-3-carboxylic acid-   TOP=Thimet oligopeptidase

Compounds of the invention are prodrug forms of therapeutic agents. Thetherapeutic agent is linked directly or indirectly to an oligopeptide ofthree amino acids, which in turn, is linked to a stabilizing group. Theprodrugs of the invention display a high specificity of action, areduced toxicity, an improved stability in serum and blood, and moveinto target cells minimally unless activated by a target cell associatedenzyme. The enzyme associated with the target cell is preferably atrouase, and more preferably is Thimet oligopeptidase or “TOP.”

Prodrug

The prodrug of the invention is a modified form of a therapeutic agentand comprises several portions, including:

(1) a therapeutic agent,

(2) an oligopeptide, and

(3) a stabilizing group, and

(4) optionally, a linker group.

Each of the portions of the prodrug are discussed in greater detailbelow. The typical orientation of these portions of the prodrug is asfollows:

(stabilizing group)-(oligopeptide)-(optional linker group)-(therapeuticagent).

The stabilizing group is directly linked to the oligopeptide at a firstattachment site of the oligopeptide. The oligopeptide is directly orindirectly linked to the therapeutic agent at a second attachment siteof the oligopeptide. If the oligopeptide and the therapeutic agent areindirectly linked, then a linker group is present.

Direct linkage of two portions of the prodrug means a covalent bondexists between the two portions. The stabilizing group and theoligopeptide are therefore directly linked via a covalent chemical bondat the first attachment site of the oligopeptide, typically theN-terminus of the oligopeptide. When the oligopeptide and thetherapeutic agent are directly linked then they are covalently bound toone another at the second attachment site of the oligopeptide. Thesecond attachment site of the oligopeptide is typically the C-terminusof the oligopeptide, but may be elsewhere on the oligopeptide.

Indirect linkage of two portions of the prodrug means each of the twoportions is covalently bound to a linker group. In an alternativeembodiment, the prodrug has indirect linkage of the oligopeptide to thetherapeutic agent. Thus, typically, the oligopeptide is covalently boundto the linker group which, in turn, is covalently bound to thetherapeutic agent.

The prodrug of the invention is cleavable within its oligopeptideportion. In order for the prodrug to be effective, the prodrug typicallyundergoes in vivo modification and an active portion, i.e., atransport-competent portion, of the prodrug enters the target cell. Afirst cleavage within the oligopeptide portion of the prodrug may leavean active or transport-competent portion of the prodrug as one of thecleavage products. Alternatively, further cleavage by one or morepeptidases may be required to result in a portion of the prodrug that iscapable of entering the cell. The active portion of the prodrug has atleast the therapeutic agent and is that part of the prodrug that canenter the target cell to exert a therapeutic effect directly or uponfurther conversion within the target cell. Thus, the compound has anactive portion, and the active portion is more capable of entering thetarget cell after cleavage by an enzyme associated with the target cellthan prior to cleavage by an enzyme associated with a target cell.

The structures of the stabilizing group and oligopeptide are selected tolimit clearance and metabolism of the prodrug by enzymes that may bepresent in blood or non-target tissue and are further selected to limittransport of the prodrug into the cells. The stabilizing group blocksdegradation of the prodrug and may act in providing preferable charge orother physical characteristics of the prodrug. The amino acid sequenceof the oligopeptide is designed to ensure specific cleavage by an enzymeassociated with a target cell, more specifically by a trouase enzyme,and even more specifically by TOP.

It is desirable to make a therapeutic agent, especially an antitumorand/or anti-inflammatory therapeutic agent, inactive by modification ofthe therapeutic agent to a prodrug form. According to the invention, thetarget cells are usually tumor cells or cells participating ininflammatory reactions, especially those associated with rheumaticdiseases, such as macrophages, neutrophils, and monocytes. Modificationof the therapeutic agent to a prodrug form also reduces some of the sideeffects of the therapeutic agents.

In the target cell, the therapeutic agent (optionally attached to one ortwo amino acids and possibly also a linker group) acts either directlyon its specific intracellular action site or, after a modification byintracellular proteases, kills the target cell or blocks itsproliferation. Since normal cells release little to no trouase in vivo,the compound according to the invention is maintained inactive and doesnot enter the normal cells or does so to a relatively minor extent.Although TOP is believed to be widely distributed in the body, it istypically present as an intracellular enzyme. Therefore it is generallyinaccessible to peptide prodrugs in the circulation. In the environmentof the tumor, TOP is believed to be released from necrotic tissue.

The prodrug is administered to the patient, carried through the bloodstream in a stable form, and when in the vicinity of a target cell, isacted upon by a trouase, such as TOP. Since the enzyme activity is onlyminimally present within the extracellular vicinity of normal cells, theprodrug is maintained and its active portion (including the therapeuticagent) gains entry into the normal cells only minimally. In the vicinityof tumor or other target cells, however, the presence of the relevantenzyme in the local environment causes cleavage of the prodrug. Theexample shown in FIG. 2 depicts an N-capped prodrug being cleavedextracellularly and the transport-competent or active portion gainingentry into the target cell. Once within the target cell, it may befurther modified to provide therapeutic effect, such as by killing thetarget cell or blocking its proliferation. While a portion of theprodrug may occasionally gain access to, and possibly harm normal cells,the transport-competent portion of the drug is freed primarily in thevicinity of target cells. Thus, toxicity to normal cells is minimized.

This process is particularly useful for, and is designed for, targetcell destruction when the target tissue releases an enzyme that is notreleased by normal cells or tissue. Here “normal cells” means non-targetcells that would be encountered by the prodrug upon administration ofthe prodrug in the manner appropriate for its intended use.

In an alternative embodiment, the orientation of the prodrug may bereversed so that the stabilizing group is attached to the C-terminus ofthe oligopeptide and the therapeutic agent is directly or indirectlylinked to the N-terminus of the oligopeptide. Thus, in the alternativeembodiment, the first attachment site may be the C-terminus of theoligopeptide and the second attachment site of the oligopeptide may bethe N-terminus of the oligopeptide. The alternative embodiment of theinvention functions in the same manner as does the primary embodiment.

In Patent Cooperation Treaty International Publication No. WO 96/05863and in U.S. Pat. No. 5,962,216, certain useful prodrugs were disclosed.The present invention is distinct from the prodrugs disclosed therein.Thus, if the oligopeptide is Leu-Ala-Leu, then the stabilizing group isnot succinyl or the therapeutic agent is not daunorubicin.

As described in greater detail below, the prodrugs of the invention arecompounds comprising:

(1) a therapeutic agent capable of entering a target cell,

(2) an oligopeptide having the formula AA³-AA²-AA¹ wherein each AAindependently represents an amino acid,

(3) a stabilizing group, and

(4) optionally, a linker group not cleavable by TOP,

wherein the oligopeptide is directly linked to the stabilizing group ata first attachment site of the oligopeptide and the oligopeptide isdirectly linked to the therapeutic agent or indirectly linked throughthe linker group to the therapeutic agent at a second attachment site ofthe oligopeptide,

wherein the stabilizing group hinders cleavage of the oligopeptide byenzymes present in whole blood, and

wherein the compound is cleaved by TOP.

Prodrugs of the invention that are cleaved by TOP under an experimentalcondition at a test rate of cleavage of 10–80% of a standard rate ofcleavage are especially useful. The standard rate of cleavage is testedon a test standard by TOP under the experimental condition, the teststandard consisting of a conjugate of Suc-βAla-Leu-Ala-Leu and thetherapeutic agent. As used herein, “cleavable by” means cleavable underphysiological conditions.

For purposes of this discussion, a compound is resistant to cleavage bya given enzyme if the rate of cleavage by a purified preparation of thegiven enzyme is no more than 15%, preferably no more than 5%, andideally no more than 1% of the rate of cleavage of Suc-βAla-Leu-Ala-Leuconjugated via the carboxyl terminus to the same therapeutic agent asthe compound of interest. The rates should be compared under the sameassay conditions. A compound is cleavable by a given enzyme if greaterthan 10% per hour, preferably greater than 50% per hour, is cleaved by amixture of the compound and the enzyme under experimental conditionswhich model physiological conditions, particularly those outside of thetarget cell. The concentration of the given enzyme in the experiment isrepresentative of the concentration of the given enzyme in theextracellular milieu of the target tissue.

Target Cell Associated Enzymes

The prodrugs of the invention are designed to take advantage ofpreferential activation through interaction with an enzyme associatedwith the target cell, at or near the site targeted within the body ofthe patient. Trouase, described in greater detail in PCT/US99/30393,incorporated herein by reference, is a class of target cell associatedenzymes.

It is believed that a trouase activates the prodrug of the invention atthe target tissue. Trouase is a class of endopeptidases that show aremarkable degree of discrimination between leucine and isoleucine atthe carboxyl side of the oligopeptide cleavage site. A definingcharacteristic is that under appropriate assay conditions, a trouasereadily cleaves Suc-βAla-Leu-Ala-Leu-Dnr while it is at leasttwenty-fold less active with Suc-βAla-Ile-Ala-Leu-Dnr.

Trouase is believed to be associated with target cells. Most likelytrouase is generated either by target cells or by normal cells that areassociated with the target cells, such as stromal tissue, neutrophils,macrophages, or B cells. So, for example, the trouase may be associatedwith or bound on (at least the active site) the outer cell surface,secreted, released or present in come manner in the extracellularvicinity of the target cell. In many cases, the prodrug of the inventionincludes a therapeutic agent for the treatment of cancer and the targetcell is a tumor cell. Thus, the trouase may be released extracellularlyby the tumor cell or it may be present extracellularly, e.g., becausecell lysis is often associated with tumors. Cell lysis is alsoassociated with inflammatory tissue, another target site.

Trouase activity is low in human plasma, however. Trouase activity hasbeen observed in carcinoma cell extracts and conditioned media fromcultured carcinoma cells, red blood cells and various human tissues,especially kidney. A partial purification scheme of trouase from HeLa(cervical carcinoma) cell homogenate ultracentrifugation (145,000×g for30 min) supernatant consists of four steps as follows:

-   -   1. Anion exchange chromatography using a 15Q column (Pharmacia)        eluted with a 0 to 0.5 M NaCl linear gradient in 20 mM        triethylamine chloride pH 7.2, 0.01% Triton X-100,    -   2. Affinity chromatography using Chelating Sepharose Fast Flow        (Pharmacia) pre-loaded with CoCl₂ and eluted with a 0 to 200 mM        imidazole linear gradient in 10 mM sodium phosphate, 0.5 M NaCl,        pH 7.2, 0.01% Triton X-100, 0.02% NaN₃,    -   3. Preparative native electrophoresis, and    -   4. Gel filtration high performance liquid chromatography using a        7.8 mm×60 cm TSK Gel G-3000SWXL (TosoHaas) column eluted with        0.3 mL/min 50 mM potassium phosphate, 200 mM potassium sulfate,        pH 7.0.

Further cleavage of the portion of the prodrug released after trouasecleavage may occur intracellularly or extracellularly, possibly byamino-exopeptidases. In vitro experiments indicate thatamino-exopeptidases of broad specificities are present in human blood aswell as the carcinoma cell environment.

Evidence now suggests that TOP is an example of a trouase. The trouaseisolated from HeLa cell extracts and studied in conditioned media orhomogenates from MCF-7/6 human carcinoma cells, catalyzes the initialcleavage of Suc-βAla-Leu-Ala-Leu-Dox. The trouase isolated from thesesources is believed to be TOP. Both structural and functional evidenceindicate that TOP is a trouase found in carcinoma cells.

According to the literature, TOP, or EC 3.4.24.15, is a thiol-activatedzinc metallopeptidase which catalyzes internal (endo) cleavage ofvarious oligopeptides having 6 to 17 amino acids (Dando, et al., “Humanthimet oligopeptidase,” Biochem J 294:451–457 (1993)). TOP is also isreferred to as Pz-peptidase, collagenase-like peptidase, kininase A,amyloidin protease, and metalloendopeptidase 24.15. The enzyme has beenisolated from chicken embryo (Morales, et al., “PZ-peptidase from chickembryos. Purification, properties, and action on collagen peptides,” JBiol Chem 252:4855–4860 (1977)), chicken liver (Barrett, et al.,“Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase,”Biochem J 271:701–706 (1990)), rat testis (Orlowski, et al.,“Endopeptidase 24.15 from rat testes. Isolation of the enzyme and itsspecificity toward synthetic and natural peptides, includingenkephalin-containing peptides,” Biochem J 261: 951–958 (1989)), andhuman erythrocytes (Dando, et al., “Human thimet oligopeptidase,”Biochem J 294:451–457 (1993)). The gene for this enzyme has been clonedand DNA sequence obtained from human brain (Dovey et al., WO 92/07068),rat testis (Pierotti, et al., “Endopeptidase-24.15 in rathypothalamic/pituitary/gonadal axis,” Mol Cell Endocrinol 76:95–103(1991)) and pig liver (Kato, et al., “Cloning, amino acid sequence andtissue distribution of porcine thimet oligopeptidase. A comparison withsoluble angiotensin-binding protein,” Eur J Biochem 221:159–165 (1994)).TOP has been immunologically or functionally identified in extracts ofHeLa (Krause, et al., “Characterization and localization ofmitochondrial oligopeptidase (MOP) (EC 3.4.24.16) activity in the humancervical adenocarcinoma cell line HeLa,” J Cell Biochem 66:297–308(1997); AT-20 cells (Crack, et al., “The association ofmetalloendopeptidase EC 3.4.24.15 at the extracellular surface of theAtT-20 cell plasma membrane,” Brain Res 835:113–124 (1999); Ferro, etal., “Secretion of metalloendopeptidase 24.15 (EC 3.4.24.15),” DNA CellBiol 18:781–789 (1999); Garrido, et al., “Confocal microscopy revealsthimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) inthe classical secretory pathway,” DNA Cell Biol 18:323–331 (1999);Madin-Darby canine kidney cells (Oliveira, et al., “Characterization ofthiol-, aspartyl-, and thiol-metallopeptidase activities in Madin-Darbycanine kidney cells,” J Cell Biochem 76 :478–488 (2000); and prostatecancer cell lines (Moody, et al., “Neurotensin is metabolized byendogenous proteases in prostate cancer cell lines,” Peptides 19:253–258(1998)).

As with TOP (Barrett, et al., “Chicken liver Pz-peptidase, athiol-dependent metallo-endopeptidase,” Biochem J 271:701–706 (1990);Lew, et al. “Substrate specificity differences between recombinant rattestes endopeptidase EC 3.4.24.15 and the native brain enzyme,” BiochemBiophys Res Commun 209: 788–795 (1995)), carcinoma cell trouase isinhibited by the metallopeptidase inhibitors EDTA and1,10-phenanthroline but not serine, thiol, or acid proteinase inhibitorssuch as aminoethylbenzene-sufonate, E64, pepstatin, leupeptin,aprotinin, CA074, or fumagillin. As reported for TOP, EDTA-treatedcarcinoma cell trouase is reactivated by Co²⁺ (50–100 μM) or Mn²⁺(50–1000 μM). Although it is also possible to reactivateEDTA-deactivated chicken (Barrett, et al., “Chicken liver Pz-peptidase,a thiol-dependent metallo-endopeptidase,” Biochem J 271 :701–706 (1990))or rat (Orlowski, et al., “Endopeptidase 24.15 from rat testes.Isolation of the enzyme and its specificity toward synthetic and naturalpeptides, including enkephalin-containing peptides,” Biochem J 261:951–958 (1989)) TOP with Zn²⁺, Zn²⁺ reactivation is not seen with theEDTA-treated MCF-7/6 cell homogenate. The specific methods used for EDTAtreatment and removal may affect the result with the carcinoma celltrouase. The fact that concentrations of Zn²⁺ as low as 100 μM areinhibitory to TOP may also be a factor. Zn²⁺ at 100 μM completelyinhibits hydrolysis of Suc-βAla-Leu-Ala-Leu-Dox by HeLa cell Fraction 1.EDTA inactivated carcinoma cell trouase can not be reactivated withcupric ions.

TOP activity is likely to be inhibited in oxygenated solutions (such asblood) and activated in mildly reducing (anoxic) environments, asdemonstrated by thiol activation of air inactivated preparations(Shrimpton et al., “Thiol activation of endopeptidase EC 3.4.24.15. Anovel mechanism for the regulation of catalytic activity,” J Biol Chem.272:17395–17399 (1997)). Accordingly, it is good candidate to selectprodrugs that are to be activated in anoxic environments such as tumortissue. Thus it is an example of a general approach for selection oftarget cell associated prodrug activating enzymes.

CD10 (CALLA, neprilysin, neutral endopeptidase, EC 3.4.24.11) is anoligopeptidase bound to the outer cell membrane of a number of cellsincluding a limited number of cancer tumor types (Turner A J (1998)Neprilysin. In Handbook of proteolytic enzymes, Barrett A J, Rawlings ND, Woessne J F (eds) pp 1108–1111. Academic Press: San Diego). Since itis also present in high concentrations in the brush boarder of theproximal kidney tubule, and at lower levels in some colon tissue and anumber of immune system cells such as B-lymphocytes it may contribute tosystemic activation of peptidyl prodrugs. This added systemic activationcould lead to increased toxicity to normal tissues when compared to apeptidyl prodrug that is not a CD10 substrate. CD10 cleaves poorly whenglycine or alanine is present in the P1′ cleavage site but cleaves wellwhen Leu is present in the P1′ site (Pozsgay et al., “Substrate andinhibitor Studies of Thermolysin-like Neutral Metalloendopeptidase FromKidney Membrane Fractions. Comparison with Bacterial Thermolysin,”Biochemistry, 25: 1292–1299 (1986)) thus Suc Leu-Ala-Gly-Dox is expectedto be poorly cleaved by CD10 compared to Suc-Leu-Ala-Leu-Dox. Thisresult is demonstrated in Example 15. When mice were given equimolardoses, the resulting plasma Dox exposure was much less with the CD10cleavable compound (Example 10) despite the fact that the rate ofhydrolysis by trouase (or TOP) of the two compounds was about the same(Example 2). As expected from the lower plasma Dox exposure, thenon-CD10 cleavable peptidyl prodrug was also much safer when tested in amean tolerated dose study (Examples 5 and 6). Thus, when treating anon-CD10 containing tumor, the preferred embodiment of this invention isa compound that is activated by TOP but not by CD10 such asSuc-Leu-Ala-Gly-Dox.

Stabilizing Group

An important portion of the prodrug is the stabilizing group, whichserves to protect the prodrug compound from cleavage in circulatingblood when it is administered to the patient and allows the prodrug toreach the vicinity of the target cell relatively intact. The stabilizinggroup typically protects the prodrug from cleavage by proteinases andpeptidases present in blood, blood serum, and normal tissue.Particularly, in the preferred embodiment, where the stabilizing groupcaps the N-terminus of the oligopeptide, and is therefore sometimesreferred to as an N-cap or N-block, the stabilizing group serves to wardagainst peptidases to which the prodrug may otherwise be susceptible.

Ideally, the stabilizing group is useful in the prodrug of the inventionif it serves to protect the prodrug from degradation, i.e., cleavage,when tested by storage of the prodrug compound in human blood at 37° C.for 2 hours and results in less than 20%, preferably less than 2%,cleavage of the prodrug by the enzymes present in the human blood underthe given assay conditions.

More particularly, the stabilizing group is either

(1) other than an amino acid, or

(2) an amino acid that is either (i) a non-genetically-encoded aminoacid having four or more carbons or (ii) aspartic acid or glutamic acidattached to the N-terminus of the oligopeptide at the β-carboxyl groupof aspartic acid or the γ-carboxyl group of glutamic acid.

For example, dicarboxylic (or a higher order carboxylic) acid or apharmaceutically acceptable salt thereof may be used as a stabilizinggroup. Since chemical radicals having more than two carboxylic acids arealso acceptable as part of the prodrug, the end group havingdicarboxylic (or higher order carboxylic) acids is an exemplary N-cap.The N-cap may thus be a monoamide derivative of a chemical radicalcontaining two or more carboxylic acids where the amide is attached ontothe amino terminus of the peptide and the remaining carboxylic acids arefree and uncoupled. For this purpose, the N-cap is preferably succinicacid, adipic acid, glutaric acid, or phthalic acid, with adipic acid andsuccinic acid being most preferred. Other examples of useful N-caps inthe prodrug compound of the invention include diglycolic acid, fumaricacid, naphthalene dicarboxylic acid, pyroglutamic acid, acetic acid, 1or 2, naphthylcarboxylic acid, 1,8-naphthyl dicarboxylic acid, aconiticacid, carboxycinnamic acid, triazole dicarboxylic acid, gluconic acid,4-carboxyphenyl boronic acid, a (PEG)_(n)-analog such as polyethyleneglycolic acid, butane disulfonic acid, maleic acid, isonipecotic acid,and nipecotic acid.

In some instances, intravascular administration of an aggregatingpositively charged prodrug in mice resulted in acute toxicity. However,no such toxicity was observed when the charge on this prodrug wasreversed by derivitization with a negatively charged stabilizing group.Many cytotoxic compounds inherently have low solubility. Positivelycharged anthracyclines for example may form aggregates at highconcentration and these aggregates may induce intravenous coagulationwhen the aggregates are administered intravenously. Since manyoligopeptides have exposed, positively-charged amino termini atphysiological pH, these aggregates may form a polypositively chargedsurface in vivo and induce a coagulation cascade within a few minutes ofadministration. This has the potential for rendering any positivelycharged prodrugs that form aggregates unsuitable for therapeutic use.

As described in greater detail in PCT/US99/30393, one way of addressingsuch a potentially dangerous obstacle is to utilize the stabilizinggroup on the peptide chain N-terminus of a negatively charged or aneutral functionality. For example, the use of succinyl as a stabilizinggroup on the prodrug alleviates the prodrug's acute toxicity. It isbelieved that the stabilizing group reduces interaction between thecompound and endothelial cell that line blood vessels. This solves animportant problem in the use of peptide prodrugs as practical therapiesfor intravenous use in humans.

Oligopeptide

Oligopeptides are generally defined as polypeptides of short length. Anoligopeptide useful in the prodrug of the invention is three amino acidsin length, however.

The oligopeptide has a formula or sequence (shown in the typicalamino-terminus to carboxy-terminus orientation) AA³-AA²-AA¹ wherein eachAA independently represents any amino acid. This corresponds to aposition sequence P1-P1′-P2′. The trouase is believed to cleave betweenthe P1 and P1′ positions, i.e., the linkage between AA³ and AA² of theoligopeptide.

The oligopeptide is written in the conventional manner with thecarboxyl-terminus (or C-terminus) at the right and the amino-terminus(or N-terminus) at the left. Thus, in the formula described, above, AA¹is the carboxyl-terminus.

In the invention of PCT/US99/30393, the oligopeptide portion of theprodrug described included a blocking, non-genetically-encoded aminoacid, as AA⁴ of the oligopeptide sequence or position P2 of the positionsequence, according to the numbering scheme described above. Thetripeptide prodrug of the present invention does not require such ablocking amino acid nor any other amino acid at position AA⁴. Despitethe short length of the oligopeptide portion of the prodrug hereindescribed, the selectivity for cleavage of the prodrug by a trouase ismaintained.

Preferred Amino Acids

Unless otherwise indicated, all amino acids are in the L configuration.Although any amino acids may be present in the oligopeptide portion ofthe prodrug, certain amino acids are preferred:

In the P1 or AA³ position, one of the following amino acids is mostpreferred: Leucine, Sarcosine, Tyrosine, Phenylalanine,p-Cl-Phenylalanine, p-Nitrophenylalanine, Valine, Norleucine, Norvaline,Phenylglycine, Tryptophan, tetrahydroisoquinoline-3-carboxylic acid,3-Pyridylalanine, Alanine, Glycine, or 2-Thienylalanine. Also preferredare Methionine or Proline in the P1 position.

In the P1′ position, AA² is most preferably selected from one of thefollowing amino acids: Alanine, Leucine, Tyrosine, Glycine, Serine,3-Pyridylalanine, or 2-Thienylalanine. Also preferred in this positionare Aminoisobutyric Acid, Threonine, or Phenylalanine.

In the P2′ position or AA¹ position, one of the following amino acids ismost preferably present: Leucine, Phenylalanine, Isoleucine, Alanine,Glycine, Tyrosine, 2-Naphthylalanine, or Serine.

Oligopeptides useful in the prodrug of the invention include thefollowing, also shown in Table 1: Leu-Ala-Leu, Tyr-Ala-Leu, Met-Ala-Leu,Tyr-Ala-Ile, Phe-Gly-Leu, Met-Gly-Leu, Met-Gly-Ile, Phe-Gly-Ile,Met-Gly-Phe, Leu-Ala-Gly, Nle-Ala-Leu, Phe-Gly-Phe, and Leu-Tyr-Leu.

TABLE 1 (AA₃) (AA₂) (AA₁) No: P1 P1′ P2′ 1 Leu Ala Leu 2 Tyr Ala Leu 3Met Ala Leu 4 Tyr Ala Ile 5 Phe Gly Leu 6 Met Gly Leu 7 Met Gly Ile 8Phe Gly Ile 9 Met Gly Phe 10  Leu Ala Gly 11  Nle Ala Leu 12  Phe GlyPhe 13  Leu Tyr LeuScreening with TOP

TOP is an important enzyme that may be utilized for selectingoligopeptides for further use and, therefore, another aspect of theinvention is an oligopeptide cleavable by TOP of the formula AA³-AA²-AA¹wherein each AA independently represents an amino acid. The oligopeptidemay be linked to a therapeutic agent and/or a stabilizing group whentesting for cleavability by TOP.

Therapeutic Agents

Therapeutic agents that are particularly advantageous to modify to aprodrug form according to the invention are those with a narrowtherapeutic window. A drug or therapeutic agent with a narrowtherapeutic window is one in which the dose at which toxicity isevident, by general medical standards, is very close to the dose atwhich efficacy is evident.

The therapeutic agent conjugated to the stabilizing group andoligopeptide and, optionally, the linker group to form the prodrug ofthe invention may be useful for treatment of cancer, inflammatorydisease, or some other medical condition. Preferably, the therapeuticagent is selected from the following classes of compounds: AlkylatingAgents, Antiproliferative agents, Tubulin Binding agents, VincaAlkaloids, Enediynes, Podophyllotoxins or Podophyllotoxin derivatives,the Pteridine family of drugs, Taxanes, Anthracyclines, Dolastatins,Topoiosomerase inhibitors, Maytanisoids, or Platinum coordinationcomplex chemotherapeutic agents.

Particularly, the therapeutic agent is advantageously selected from thefollowing compounds: or a derivative or analog thereof Doxorubicin,Daunorubicin, Viniblastine, Vincristine, Calicheamicin, Etoposide,Etoposide phosphate, CC-1065, Duocarmycin, KW-2189, Methotrexate,Methopterin, Aminopterin, Dichloromethotrexate, Docetaxel, Paclitaxel,Epithiolone, Combretastatin, Combretastatin A₄ Phosphate, Dolastatin 10,Dolastatin 11, Dolastatin 15, Topotecan, Camptothecin, Mitomycin C,Porfiromycin, 5-Fluorouracil, 6-Mercaptopurine, Fludarabine, Tamoxifen,Cytosine arabinoside, Adenosine Arabinoside, Colchicine, Carboplatin,cis-Platin, Maytansine, Mitomycin C, Bleomycin, Melphalan, Chloroquine,or Cyclosporin A. By derivative is intended a compound that results fromreacting the named compound with another chemical moiety, and includes apharmaceutically acceptable salt, acid, base or ester of the namedcompound. By analog is intended a compound having similar structural andfunctional properties, such as biological activities, to the namedcompound.

Linker Groups

A linker group between the oligopeptide and the therapeutic agent may beadvantageous for reasons such as the following:

-   -   1. As a spacer for steric considerations in order to facilitate        enzymatic release of the AA¹ amino acid or other enzymatic        activation steps.    -   2. To provide an appropriate attachment chemistry between the        therapeutic agent and the oligopeptide.    -   3. To improve the synthetic process of making the prodrug        conjugate (e.g., by pre-derivitizing the therapeutic agent or        oligopeptide with the linker group before conjugation to enhance        yield or specificity.)    -   4. To improve physical properties of the prodrug.    -   5. To provide an additional mechanism for intracellular release        of the drug.

Linker structures are dictated by the required functionality. Examplesof potential linker chemistries are hydrazide, ester, ether, andsulphydryl. Amino caproic acid is an example of a bifunctional linkergroup. When amino caproic acid is used in the linker group, it is notcounted as an amino acid in the numbering scheme of the oligopeptide.

The optionally present linker group is not cleavable by TOP, i.e. it isnot cleavable by TOP under physiological conditions.

Screening of the Prodrug

As mentioned previously, the synthesized prodrug ideally should betested against a test standard which consists of Suc-βAla-Leu-Ala-Leuconjugated to a therapeutic agent or marker. The same therapeutic agentor marker that was used to make the prodrug compound should beconjugated to the Suc-βAla-Leu-Ala-Leu to make the test standard. Therates of hydrolysis of the synthesized tripeptide prodrug and the teststandard by a trouase are compared under common experimental conditions.Example 2 below provides an exemplary scheme for performing this test.The tripeptide prodrugs of the invention are preferably cleaved at arate that is a fractional portion of the standard rate of cleavage forthe test standard. The most useful prodrugs of the present inventioncleave at a fractional portion equal to 10–80% of the rate of cleavageof the test standard. Even more preferably, a prodrug of the presentinvention cleaves at a rate that is 30–65% of the rate of cleavage ofthe test standard.

The disclosure of making and using tripeptide prodrugs taught hereinprovides a useful alternative to prior teachings of prodrug design. Asillustrated in the examples below, the prodrugs of the invention areefficacious and well-tolerated in vivo in animal models. As such, theprodrugs are advantageously utilized in therapy.

An especially useful embodiment is a compound that is cleavable by atrouase but resistant to cleavage by CD10 or other systemic or bloodenzymes.

Prodrug Design

A method of designing a prodrug is another aspect of the invention andentails initially selecting an oligopeptide of three amino acids. Thenthe oligopeptide is linked at a first attachment site of theoligopeptide to a stabilizing group that hinders cleavage of theoligopeptide by enzymes present in whole blood, and directly orindirectly linked to a therapeutic agent at a second attachment site ofthe oligopeptide. The linkage of the oligopeptide to the therapeuticagent and the stabilizing group may be performed in any order orconcurrently. The resulting conjugate is tested for cleavability by atrouase, such as TOP. Preferably, the resulting conjugate is tested forcleavage under a given experimental condition, and the conjugate isselected as a prodrug if the test rate of cleavage is 10–80%, or morepreferably 30–65%, of a standard rate of cleavage. The standard rate ofcleavage is tested on a test standard by the trouase under the samegiven experimental condition. The test standard consists of a conjugateof Suc-βAla-Leu-Ala-Leu and the therapeutic agent (or the marker, in thecase of the article of manufacture described below). The resultingconjugate may also be tested for stability in whole blood. Testcompounds stable in whole blood are selected. The first attachment siteis usually the N-terminus of the oligopeptide but may be the C-terminusof the oligopeptide or another part of the oligopeptide. The secondattachment site is usually the C-terminus of the oligopeptide, but maybe the N-terminus of the oligopeptide or another part of theoligopeptide. A prodrug designed by such a method is also part of theinvention.

Further, the invention includes a method for decreasing toxicity of atherapeutic agent that is intended for administration to a patient.Specifically, a modified, prodrug form of the therapeutic agent isformed by directly or indirectly linking the therapeutic agent to anoligopeptide of three amino acids that is cleavable by a trouase, ormore specifically, cleavable by TOP. The oligopeptide is also linked toa stabilizing group. The prodrug thus formed should be selectivelycleaved by the trouase at a test rate of cleavage that is 10–80%, ormore preferably 30–65%, of a standard rate of cleavage resulting fromthe test standard, as described. The prodrug provides for decreasedtoxicity of the therapeutic agent when administered to the patient. Themodification of the therapeutic agent in this manner also allows foradministration of an increased dosage of the therapeutic agent to thepatient relative to the dosage of the therapeutic agent in unconjugatedform.

Pharmaceutical Compositions

The invention also includes a pharmaceutical composition comprising acompound, particularly a prodrug compound, according to the inventionand, optionally, a pharmaceutically acceptable carrier, for example anadjuvant, vehicle, or the like.

The invention also relates to the use of the pharmaceutical compositionfor the preparation of a medicinal product intended for the treatment ofa medical condition.

The pharmaceutical composition may, for example, be administered to thepatient parenterally, especially intravenously, intramuscularly, orintraperitoneally. Pharmaceutical compositions of the invention forparenteral administration comprise sterile, aqueous or nonaqueoussolutions, suspensions, or emulsions. As a pharmaceutically acceptablesolvent or vehicle, propylene glycol, polyethylene glycol, injectableorganic esters, for example ethyl oleate, or cyclodextrins may beemployed. Isotonic saline may be part of the pharmaceutical composition.These compositions can also comprise wetting, emulsifying and/ordispersing agents.

The sterilization may be carried out in several ways, for example usinga bacteriological filter, by incorporating sterilizing agents in thecomposition or by irradiation. They may also be prepared in the form ofsterile solid compositions, which may be dissolved at the time of use insterile water or any other sterile injectable medium.

The pharmaceutical composition may also comprise adjuvants that are wellknown in the art (e.g., vitamin C, antioxidant agents, etc.) and capableof being used in combination with the compound of the invention in orderto improve and prolong the treatment of the medical condition for whichthey are administered.

Doses for administration to a patient of the compounds according to theinvention are generally at least the usual doses of the therapeuticagents known in the field, described in Bruce A. Chabner and Jerry M.Collins, Cancer Chemotherapy, Lippincott Ed., ISBN 0-397-50900-6 (1990)or they may be adjusted, within the judgment of the treating physician,to accommodate the superior effectiveness of the prodrug formulations orthe particular circumstances of the patient being treated. Hence, thedoses administered vary in accordance with the therapeutic agent usedfor the preparation of the compound according to the invention.

Treatment of Patients with Prodrug Compound

A method for the therapeutic treatment of a medical condition thatinvolves administering, preferably parenterally and more preferablyintravenously, to the patient a therapeutically effective dose of thepharmaceutical composition is also within the scope of the invention.Thus, the method generally entails administering to the patient atherapeutically effective amount of a compound comprising:

(1) a therapeutic agent capable of entering a target cell,

(2) an oligopeptide of the formula AA³-AA²-AA¹ wherein each AAindependently represents an amino acid,

(3) a stabilizing group, and

(4) optionally, a linker group not cleavable by a trouase,

-   -   wherein the oligopeptide is directly linked to the stabilizing        group at a first attachment site of the oligopeptide and the        oligopeptide is directly linked to the therapeutic agent or        indirectly linked through the linker group to the therapeutic        agent at a second attachment site of the oligopeptide,    -   wherein the stabilizing group hinders cleavage of the        oligopeptide by enzymes present in whole blood, and    -   wherein the compound is selectively cleaved by the trouase,        preferably TOP. The most useful compounds result in a test rate        of cleavage of 10–80% of a standard rate of cleavage under a        given experimental condition. The standard rate of cleavage is        tested on a test standard by the trouase under the same        experimental condition and the test standard consists of a        conjugate of Suc-βAla-Leu-Ala-Leu and the therapeutic agent.        More preferably, the test rate of cleavage of the compound is        30–65% of the standard rate of cleavage.

The prodrug compound is useful for the treatment of many medicalconditions including cancer, neoplastic diseases, tumors, inflammatorydiseases, and infectious diseases. Examples of preferred diseases arebreast cancer, colorectal cancer, liver cancer, lung cancer, prostatecancer, ovarian cancer, brain cancer, and pancreatic cancer. Formulatedin pharmaceutically acceptable vehicles (such as isotonic saline), theprodrug compound can be administered to animals or humans in intravenousdoses ranging from 0.05 mg/kg/dose/day to 300 mg/kg/dose/day. It canalso be administered via intravenous drip or other slow infusion method.

Human patients are the usual recipients of the prodrug of the invention,although veterinary usage is also contemplated.

Diagnosis or Assay

An article of manufacture, such as a kit, for diagnosis or assay is alsowithin the scope of the invention. Such an article of manufacture wouldpreferably utilize a compound as described above, except that a marker,such as coumarin, is conjugated to the oligopeptide and stabilizinggroup instead of a therapeutic agent. A marker intends any moiety thatcan be conjugated to the oligopeptide and is readily detectable by anymethod known in the art. At least one reagent useful in the detection ofthe marker is typically included as part of the kit. Thus, the articleof manufacture would include a compound and optionally a least onereagent useful in the detection of a marker. More particularly, thecompound comprises:

(a) a marker,

(b) an oligopeptide of the formula AA³-AA²-AA¹ wherein each AAindependently represents an amino acid,

(e) a stabilizing group, and

(d) optionally, a linker group not cleavable by TOP or another trouase,

-   -   wherein the oligopeptide is directly linked to the stabilizing        group at a first attachment site of the oligopeptide and the        oligopeptide is directly linked to the marker or indirectly        linked through the linker group to the marker at a second        attachment site of the oligopeptide,    -   wherein the stabilizing group hinders cleavage of the        oligopeptide by enzymes present in whole blood, and    -   wherein the compound is selectively cleaved by the trouase. As        before, most useful are those compounds that cleave at a test        rate of cleavage of 10–80% of a standard rate of cleavage under        an experimental condition, the standard rate of cleavage tested        on a test standard by the trouase under the same experimental        condition, the test standard consisting of a conjugate of        Suc-βAla-Leu-Ala-Leu and the marker. The article of manufacture        may be used, for example, with patient samples to diagnose        tumors or to identify patients susceptible to treatment by        prodrug therapy.        Process Chemistry General Procedures        Oligopeptide: General Method for the Synthesis of Peptides

The peptide, or oligopeptide, sequences in the prodrug conjugates ofthis invention may be synthesized by the solid phase peptide synthesis(using either Boc or Fmoc chemistry) methods or by solution phasesynthesis. The general Boc and Fmoc methods are widely used and aredescribed in the following references: Merrifield, J. A. Chem. Soc.,88:2149 (1963); Bodanszky and Bodanszky, The Practice of PeptideSynthesis, Springer-Verlag, Berlin, 7–161 (1994); Stewart, Solid PhasePeptide Synthesis, Pierce Chemical, Rockford, (1984).

General Fmoc Solid Phase Method

Using the preferred solid phase synthesis method, either automated ormanual, a peptide of desired length and sequence is synthesized throughthe stepwise addition of amino acids to a growing chain which is linkedto a solid resin. Examples of useful Fmoc compatible resins include, butare not limited to, Wang resin, HMPA-PEGA resin, Rink acid resin, or ahydroxyethyl-photolinker resin. The C-terminus of the peptide chain iscovalently linked to a polymeric resin and protected α-amino acids wereadded in a stepwise manner with a coupling reagent. A preferred α-aminoprotecting group is the Fmoc group, which is stable to couplingconditions and can readily be removed under mild alkaline conditions.The reaction solvents are preferably but not limited to DMF, NMP, DCM,MeOH, and EtOH. Examples of coupling agents are: DCC, DIC, HATU, HBTU.Cleavage of the N-terminal protecting group is accomplished in 10–100%piperidine in DMF at 0–40° C., with ambient temperature being preferred.At the end of synthesis, the final Fmoc protecting group is removedusing the above N-terminal cleavage procedure. The remaining peptide onresin is cleaved from the resin along with any acid sensitive side chainprotecting groups by treating the resin under acidic conditions. Forexample, an acidic cleavage condition is a mixture of trifluoroaceticacid (TFA) in dichloromethane. If the hydroxyethyl-photolinker resin isused, the appropriate wavelength for inducing cleavage is λ 365 nmultraviolet light. A diagramatic representation of this process is givenin FIG. 3.

General N-Cap Method via Solid Phase Synthesis

The preparation of N-terminus derivatized peptides is convenientlyaccomplished on solid phase. When the peptide synthesis is complete, theterminal Fmoc is removed while the peptide is still on the solidsupport. The N-cap of choice is coupled next using standard peptidecoupling conditions onto the N-terminus of the peptide. On completion ofthe N-cap coupling the peptide is cleaved from the resin using theprocedure described above.

General Boc Solid Phase Method

For the solid phase method using Boc chemistry, either the Merrifieldresin or PAM resin is useful. The amino acids are coupled to the growingchain on solid phase by successive additions of coupling agent activatedBoc-protected amino acids. Examples of coupling agents are: DCC, DIC,HATU, HBTU. The reaction solvents may be DMF, DCM, MeOH, and NMP.Cleavage of the Boc protecting group is accomplished in 10–100% TFA inDCM at 0–40° C., with ambient temperature being preferred. On completionof the peptide chain assembly the N-terminus protecting group (usuallyBoc) is removed as described above. The peptide is removed from theresin using liquid HF or trifluoromethane sulfonic acid indichloromethane.

General Procedure for the Preparation of Fmoc Oligopeptide by SolutionPhase Synthesis

Alternatively, the prodrug peptide intermediate may be made via asolution phase synthesis, utilizing either Boc or Fmoc chemistry. In thediagrammatic presentation of the methods (FIG. 4), the C-terminal Leutripeptide is generally used as an example, but it will be understoodthat similar reactions may be performed with other C-terminaltripeptides, as well. The peptide can be built up by the stepwiseassembly in analogy to the solid phase method (in the N-terminaldirection or in the C-terminal direction) or through the coupling of adipeptide with a single amino acid.

One method of solution phase synthesis is a stepwise building up of theprodrug peptide intermediate using Fmoc chemistry, shown in FIG. 4. TheC-terminus must be protected to reduce the formation of side products.The C-terminal R group in FIG. 4 is Me, tBu, benzyl or TCE. (Note whenthe N-cap is methyl succinyl the C-terminus R group cannot be methyl.)Although DMF is given as the solvent, other solvents such as DMSO,CH₃CN, or NMP (or mixtures thereof) may be substituted therefor.Pyridine, Et₃N or other bases may be substituted for piperidine indeprotecting the growing peptide chain protected amino terminus.Similarly, although HBTU is given in the diagram above as the activatingagent, other activating agents such as DCC, DIC, DCC+HOBt, OSu,activated esters, azide, or triphenyl phosphoryl azide may be used.Additionally, the protected peptide acid chloride or acid bromide may beused to couple directly to the amino acid or peptide fragment. Oncompletion of the oligopeptide assembly, the N-terminus is deprotectedand the C-terminus protected peptide is ready to accept the desiredN-cap.

General Procedure for the Preparation of N-Cap Oligopeptide via SolutionPhase Synthesis

When constructing the N-capped oligopeptide by solution phase synthesis,the N-cap needs to be synthesized by a slightly modified procedure (FIG.5). First the C-terminus of the Fmoc oligopeptide needs to be protectedwith an acid labile or hydrogenation sensitive protecting groupcompatible with the selective deprotection of the C-terminus over theN-cap. Then the Fmoc protecting group needs to be removed from theoligopeptide to reveal the N-terminus. With the N-terminus deprotectedand the C-terminus protected, the oligopeptide is reacted with theactivated hemiester of the desired N-cap. The N-cap can be activatedusing methods for activating amino acids such as DCC or HATU in base andan appropriate solvent. Alternatively, where the methyl-hemisuccinate isused, the coupling may also be done via methyl hemisuccinyl chloride (orother acid halide) (FIG. 4) using an inert solvent in the presence of anorganic or inorganic base, such as DIEA, triethylamine or Cs₂CO₃. Oneexample of such a synthesis can be by reacting methylhemisuccinate andMet-Ala-Leu benzyl ester. The coupling method can be any one of themethods generally used in the art (see for example: Bodanszky, M., ThePractice of Peptide Synthesis, Springer Verlag, 185 (1984); Bodanszky,M., Principles of Peptide Synthesis, Springer Verlag, 159 (1984). Thebenzyl group then can be removed by catalytic hydrogenation providingthe desired N-cap methylsuccinyl form of the oligopeptide. Otherexamples of suitable, selectively removable C-terminal protecting groupscan be, but are not limited to, tBu, alkoxy-methyl and TCE. Othermethods of accomplishing this step are described in the literature.

The reaction conditions are well known in the art and detailed in thecitations given. The advantage of the above described methods is thefacile purification of the product produced by solution phase synthesis.

Prodrug Conjugate

General Methods for the Conjugation and Deprotection Steps

The prodrugs described herein can be synthesized by coupling an Fmocform (which means Fmoc is attached to the N-terminus of theoligopeptide) of the oligopeptide with daunorubicin, or doxorubicin, orany appropriate therapeutic agent using any of the standard activatingreagents used in peptide synthesis (FIG. 5). The solvent may be toluene,ethyl acetate, DMF, DMSO, CH₃CN, NMP, THF, DCM or any other suitableinert solvent as is known in the art and the reagents are solubletherein. The preferred solvents are DMF and NMP. The appropriatetemperature range is −25 to +25° C., with ambient temperature beingpreferred. The activating agent may be selected from one of thefollowing: PyBOP, HBTU, HATU, EDC, DIC, DCC, DCC+HOBT, OSu activatedesters, azide, or triphenylphosphorylazide. HBTU or HATU is thepreferred activating agent. Alternatively, the acid chloride or the acidbromide of the protected peptide can also be used for this couplingreaction. 2–4 equivalents, advantageously 2–2.5 equivalents of a base isrequired for the coupling reaction. The base can be selected frominorganic bases such as CsCO₃, Na- or K₂CO₃, or organic bases, such asTEA, DIEA, DBU, DBN, DBO, pyridine, substituted pyridines,N-methyl-morpholine etc., preferably TEA, or DIEA. The reaction can becarried out at temperatures between −15° C. and 50° C., advantageouslybetween −10° C. and 10° C. The reaction time is between 5–90 minutes andis advantageously 20–40 minutes. The product is isolated by pouring thereaction mixture into water and filtering the precipitate formed. Thecrude product can be further purified by recrystallization from DCM,THF, ethyl acetate, or acetonitrile, preferably from dichloromethane oracetonitrile. The isolated Fmoc form of the (oligopeptide)-(therapeuticagent) conjugate is then deprotected over 2–90 minutes, preferably 3–8minutes, using a ten- to hundred-fold excess of base at a temperaturebetween −10° C. and 50° C. Ideally, 5–60 equivalents of the base arepreferred. Piperidine is the preferred base to deprotect Fmoc groups.The deprotected amino terminus of the (oligopeptide)-(therapeutic agent)conjugate is acylated by a diacid anhydride as an activated hemiester togive the final N-cap form of the oligopeptide-therapeutic agent.

Alternatively, the final prodrug can be similarly prepared from theprotected N-cap form of the oligopeptide such as a methylhemiester formof succinyl-N-cap oligopeptide and conjugated to a therapeutic agent.This method is illustrated in FIG. 6.

The (protected N-Cap)-(oligopeptide)-(therapeutic agent) conjugate isnow deprotected by methods compatible to the stability of thetherapeutic agent. For example, anthracyclines may be protected with amethyl group and deprotected with an esterase. For other therapeuticagents we might select benzyl protecting groups and catalytichydrogenation may be chosen to deprotect.

Conversion to the salt form of the negatively charged(N-cap)-(oligopeptide)-(therapeutic agent) is carried out with a solventselected from the following group: alcohol (including methanol, ethanol,or isopropanol), water, acetonitrile, tetrahydrofuran, diglyme or otherpolar solvents. The sodium source is one molar equivalent of NaHCO₃,NaOH, Na₂CO₃, NaOAc, NaOCH₃ (in general sodium alkoxide), or NaH. An ionexchange column charged with Na⁺ (such as strong or weak ion exchangers)is also useful for this last step of making the salt form of the(N-cap)-(oligopeptide)-(therapeutic agent) when appropriate. Sodium isdescribed in this application as an example only.

Generally, the prodrug may be converted to a pharmaceutically acceptablesalt form to improve solubility of the prodrug. The(N-cap)-(oligopeptide)-(therapeutic agent) is neutralized with apharmaceutically acceptable salt, e.g., NaHCO₃, Na₂CO₃, NaOHtris(hydroxymethyl)aminomethane, KHCO₃, K₂CO₃, CaCO₃, NH₄OH, CH₃NH₂,(CH₃)₂NH, (CH₃)₃N, acetyltriethylammonium. The preferred salt form ofprodrug is sodium and the preferred neutralizing salt is NaHCO₃.

It is well documented that anthracycline type molecules, includingdoxorubicin and daunorubicin form gels in organic solvents in very lowconcentrations (Matzanke, B. F., et al., Eur. J. Biochem., 207:747–55(1992); Chaires, J. B., et al., Biochemistry, 21:3927–32 (1982);Hayakawa, E., et al., Chem. Pharm. Bull., 39:1282–6 (1991). This may bea considerable obstacle to getting high yields of clean product whenmaking peptide anthracycline conjugates. The gel formation contributesto the formation of undesirable side reactions. One way to minimize thisproblem is to use very dilute solutions (1–2%) for the couplingreaction, however it is not practical in a process environment (largeamounts of waste, complicated isolation). To overcome this problem, ureaor other chaotropic agents may be used to break up the stronghydrophobic and hydrogen bonding forces forming the gel. Thus if thecoupling reaction is carried out in a urea-containing solvent,advantageously a 20% to saturated solution of urea in DMF or NMP, theside reactions can be kept below 2% even if the concentration ofreactants exceeds 10%. This procedure makes the conjugation steppractical at high concentrations and produces good yields.

General Enzyme Method

Hydrolysis of protected N-cap-oligopeptide therapeutic agents to thefull N-cap compound catalyzed by acids or bases leads to complexreaction mixtures due to the lability of many therapeutic agents evenunder moderately acidic or basic conditions. Enzymes can promote thehydrolysis without destroying the substrate or the product. Enzymessuitable for this reaction can be esterases or lipases and can be intheir natural, water soluble forms or immobilized by cross coupling, orattachment to commercially available solid support materials. Of thesoluble enzymes evaluated, Candida Antarctica “B” lipase (AltusBiologics) is especially useful. An example of an enzyme immobilized bycross coupling is ChiroCLEC-PC™ (Altus Biologics). Candida Antarctica“B” lipase (Altus Biologics) can be immobilized by reaction with NHSactivated Sepharose™ 4 Fast Flow (American Pharmacia Biotech). The pH ofthe reaction mixture during the hydrolysis is carefully controlled andmaintained by a pH-stat between 5.5 and 7.5, advantageously between 5.7and 6.5, via controlled addition of NaHCO₃ solution. When the reactionis completed the product is isolated by lyophilization of the filteredreaction mixture. The immobilized enzymes remain on the filter cake andcan be reused if desired.

General Allyl or Alkyl Ester Method

The prodrug can also be prepared via coupling an allyl-hemiester oralkyl-hemiester form of the N-cap oligopeptide with a therapeutic agentand then liberating the free acid from the conjugate. FIG. 8 illustratesthis process with Succinyl-Met-Ala-Leu and doxorubicin.

The coupling of allyl-succinyl-Met-Ala-Leu with doxorubicin can becarried out via any one of the oligopeptide conjugation methods.

Allyl-succinyl-Met-Ala-Leu-doxorubicin can also be synthesized byreacting allyl hemisuccinate, which was prepared via known methods(Casimir, J. R., et al., Tet. Lett. 36/19 3409 (1995)), withMet-Ala-Leu-doxorubicin similarly as coupling of the protectedtripeptide precursors to doxorubicin was described in the previousmethods, shown in FIG. 5. Suitable inert solvents are THF,dichloromethane, ethyl acetate, toluene, preferably THF from which theacid form of the product precipitates as the reaction progresses. Theisolated acid is converted to its sodium salt as described earlier.Reaction times vary between 10–180 minutes, advantageously 10–60minutes, at temperatures between 0–60° C., preferably 15–30° C.

Removal of the allyl or alkyl group can be done with Pd (0), or Ni(0),advantageously Pd(0) promoted transfer of the allyl or alkyl group toacceptor molecules, as it is well known in the art and documented in theprofessional literature (Genet, J-P, et al., Tet. Lett., 50, 497, 1994;Bricout, H., et.al. Tet. Lett., 54:1073 (1998), Genet, J-P. et.al.Synlett, 680 (1993); Waldmann, H., et.al., Bioorg. Med. Chem., 7:749(1998); Shaphiro, G., Buechler, D., Tet. Lett., 35:5421 (1994)). Theamount of catalyst can be 0.5–25 mol % to the substrate.

General Trityl or Substituted Trityl Method

The prodrug may also be synthesized via the method shown in FIG. 7. Thisapproach utilizes an R′-oligopeptide, where R′ is trityl or substitutedtrityl. The coupling of R′-oligopeptide with a therapeutic agent can becarried out via any one of the methods described earlier for conjugationof a protected oligopeptide with a therapeutic agent at 30–120 minutesat 0–20° C.

Removal of trityl or substituted trityl group can be achieved underacidic conditions to give the positively charged prodrug. Thispositively charged prodrug is N-capped as illustrated in FIG. 4 anddescribed earlier. The trityl deprotection can be accomplished withacetic acid, formic acid and dilute hydrochloric acid.

The prodrug can be converted into (succinyl orglutaryl)-(oligopeptide)-(therapeutic agent) conjugate by reacting withsuccinic anhydride or glutaric anhydride. The solvent for coupling stepDMF, DMSO, CH₃CN, NMP, or any other suitable solvent is known in theart. Succinyl or glutaryl oligopeptide therapeutic agents can beconverted to any pharmaceutically acceptable salt.

General Inverse Direction Solid Phase Conjugation Method

The prodrug compound of the present invention can be synthesized byusing solid phase chemistry via “step wise” inverse (from the N-terminalto the C-terminal) direction methods.

One way is to use resins to immobilize a succinyl hemiester, for examplesuccinyl-mono-benzyl ester or -allyl ester. Examples of resins could beselected are “Wang Resins” (Wang, S. S., J. Am. Chem. Soc., 95:1328(1973); Zhang, C., Mjaili, A. M. M., Tet. Lett., 37:5457(1996)), “RinkResins” (Rink, H., Tet. Lett., 28:3787 (1987)), “Trityl-, orsubstituted-trityl Resins” (Chen, C., et.al., J. Am. Chem. Soc.,116:2661 (1994); Bartos, K. et.al., Peptides, Proc. 22^(nd) EuropeanPeptide Symposium (1992); Schneider, C. H.; Eberle, A. N. (Eds.), ESCOM,Leiden, pp. 281 (1993). The immobilized ester is then deprotected andreacted with, for example, a similarly C-terminal protected methionine.These steps are then repeated with alanine, and finally leucine esters,followed by the coupling of doxorubicin to the immobilizedsuccinyl-tripeptide. The molecule is then liberated from the resin byusing mildly acidic conditions to form a free prodrug, such as freeSuc-Met-Ala-Leu-Dox. This methodology is represented on the scheme ofFIG. 9. Another version of phase synthesis utilizes immobilized succinyloligopeptide ester. This is then C-terminally deprotected, followed bythe coupling step to doxorubicin or other therapeutic agent, and finallyliberated from the resin as represented on the scheme of FIG. 9. Theacid form of the prodrug molecule may then be converted finally into itssodium salt as described above.

General Large Scale Compound Synthesis

The prodrug compound can be synthesized using a simple and efficientthree-step process of the invention: (1) coupling an alkyl or allylester protected stabilizing group-oligopeptide and a therapeutic agentin the presence of an activating agent to make an alkyl or allyl esterprotected stabilizing group-oligopeptide-therapeutic agent conjugate,(2) removing uncoupled therapeutic agent that remains after the couplingstep, and (3) deprotecting the alkyl or allyl ester protectedstabilizing group-oligopeptide-therapeutic agent conjugate to make thestabilizing group-oligopeptide-therapeutic agent prodrug compound.

The first step involves the coupling of an alkyl-ester protectedoligopeptide fragment to a therapeutic agent. A preferred embodiment ofthe first step involves the coupling of an alkyl or allyl esterprotected stabilizing group oligopeptide, such as MeOSuc-Leu-Ala-Leu-OH,with a therapeutic agent, such as doxorubicin, using an activatingagent, such as HATU, to give an alkyl or allyl ester protectedstabilizing group oligopeptide therapeutic agent conjugate, e.g.,MeOSuc-Leu-Ala-Leu-Dox. The focus of this step is on the purity and theyield of the methyl ester, since it was found that the hydrolysis stepdoes not have a significant impact on purity. Preferably the molar ratioof the alkyl or allyl ester protected stabilizing group oligopeptide tothe therapeutic agent will be between 2:1 and 1:1. More preferably themolar ratio is between 1.75:1 and 1.5:1. Most preferably the molar ratiois 1.66:1.

The coupling of the alkyl or allyl ester protected stabilizing groupoligopeptide and a therapeutic agent is preferably performed by: (a)combining the alkyl or allyl ester protected stabilizing groupoligopeptide and the therapeutic agent in DMF, (b) adding DIEA, (c)reacting the alkyl or allyl ester protected stabilizing groupoligopeptide and the therapeutic agent in the presence of the activatingagent to form the conjugate, and (d) precipitating the conjugate byadding a brine solution to form a precipitate. Preferably the molarratio of the DIEA and the alkyl or allyl ester protected stabilizinggroup-oligopeptide is between 3:1 and 1.5:1. More preferably the molarratio is 2.5:1 and 2:1. Most preferably the molar ratio is 2.18:1. Thereacting step is preferably performed at 0° C., for 30 minutes.Preferably the molar ratio of the activating agent and the alkyl orallyl ester protected stabilizing group-oligopeptide is between 1.5:1and 1:1. More preferably, the molar ratio is 1.1:1. The brine solutionis preferably between 20% (w/v) and 40% (w/v) of NaCl in water. Morepreferably the brine solution is preferably between 25% (w/v) and 35%(w/v) of NaCl in water. Most preferably the brine solution is 30% (w/v)of NaCl in water. The conjugate is preferably precipitated in a brinesolution, wherein the pH is between 5.0 and 7.0, inclusive. Mostpreferably, the conjugate is precipitated at a pH between 5.8 and 6.0.

Since many therapeutic agents are toxic substances, it is preferable toeliminate any free therapeutic agent from the coupled product. Theremoving step is preferably performed by: (a) dissolving the conjugatein DMF, (b) dissolving a scavenger resin in anhydrous DMF, (c) addingthe alkyl or allyl ester protected stabilizing group oligopeptidetherapeutic agent conjugate formed in the coupling step to the scavengerresin to form a conjugate-resin mixture, (d) maintaining the mixture atbetween 0° C. and 30° C. for 2 to 24 hours wherein the uncoupledtherapeutic agent reacts with the resin, (e) removing the resin from themixture, and (f) precipitating the remainder by adding a brine solutionto form a precipitate of the alkyl or allyl ester protected stabilizinggroup oligopeptide therapeutic agent conjugate. Preferably the scavengerresin is polystyrene-isocyanate (PS-isocyanate), PS-methylisocyanate,PS-thioisocyanate, PS-methylthioisocyanate, PS-sulfonyl chloride,PS-methylsulfonyl chloride or PS-benzaldehyde. Most preferably, thescavenger resin is PS-isocyanate. The removing step is preferablyperformed to remove free therapeutic agent, which is an anthracycline.

The third step is deprotecting the alkyl or allyl ester protectedstabilizing group-oligopeptide-therapeutic agent conjugate, preferablyvia hydrolysis by an enzyme, more preferably via hydrolysis by anesterase, which directly gives the prodrug compound in good yield with afinal purity of at least 90%. For example, the third step may be thehydrolysis of the methyl ester group in MeOSuc-Leu-Ala-Leu-Dox by anenzyme, such as CLEC CAB (crosslinked Candida Antartica B Lipase), whichdirectly gives the sodium salt of Suc-Leu-Ala-Leu-Dox in quantitativeyields with high purity.

The enzyme is preferably either crosslinked or immobilized on a solidsupport. The esterase may be pig liver esterase, Candida Antartica BLipase, Candida Rugosa lipase, Pseudomonas Cepacia lipase, pig liveresterase immobilized on sepharose, Candida antartica B lipaseimmobilized on sepharose, CLEC-PCTM (Pseudomonas Cepacia lipase),CLEC-CAB (Candida Antartica B lipase), or CLEC-CR (Candida Rugosalipase). Deprotecting via hydrolysis by an enzyme is preferablyperformed by: (a) washing the enzyme to remove free enzyme, (b) addingthe washed enzyme to the alkyl or allyl ester protected stabilizinggroup-oligopeptide-therapeutic agent conjugate, (c) reacting the enzymewith the conjugate at between 15° C. and 40° C., inclusive, at a pHbetween 5.0 and 8.0, inclusive, for at least 18 hours, to create thestabilizing group-oligopeptide-therapeutic agent prodrug compound, and(d) separating the enzyme from the prodrug compound. Most preferablyadditional washed crosslinked or immobilized enzyme is added after thestep of reacting the enzyme with the conjugate, prior to separating theenzyme from the prodrug compound.

Removal of Free Therapeutic Agent

Unconjugated therapeutic agent may be present late in the process ofmaking the prodrug. For example, during the coupling step of(stabilizing group)-(oligopeptide) conjugate with doxorubicin as thetherapeutic agent, it was found, in some instances, that the reactiondid not proceed completely. There was about 2–4% of residual doxorubicinremaining in the coupled product. Initial attempts to remove doxorubicincompletely from the product by acidic washes did not result in completeremoval. The complete removal of the free therapeutic agent was effectedby the process outlined in Example 29 and FIG. 17 that utilizesscavenging resin or beads.

The crude product, which contains the intermediate and residualdoxorubicin, were dissolved in DMF and polystyrene methylisocyanate orpolystyrene sulfonyl chloride resin or beads were added. The reactionwas stirred for 60 minutes. The free amino group of doxorubicin reactswith the isocyanate or sulfonyl chloride group on the beads to form aurea or sulfonamide derivative. The solid beads with doxorubicinattached to them were then separated from the desired product byfiltration. The desired product remains in the DMF solution. Thisapproach seems to be a very mild and effective method for removingresidual therapeutic agent from the product.

Thus, the invention includes a method of making a compound comprising:

-   (1) selecting an Fmoc-protected oligopeptide of the formula    Fmoc-AA³-AA²-AA¹ wherein each AA independently represents an amino    acid,-   (2) coupling the Fmoc-protected oligopeptide to a therapeutic agent    by activating the Fmoc-protected oligopeptide with an activating    agent in the presence of the therapeutic agent to form an    Fmoc-protected oligopeptide-therapeutic agent conjugate,-   (3) deprotecting the Fmoc-protected oligopeptide-therapeutic agent    conjugate by contacting it with a base to form an    oligopeptide-therapeutic agent conjugate, and-   (4) coupling the oligopeptide-therapeutic agent conjugate to a    stabilizing group to form the compound.

Alternatively, a method of making a compound comprises the followingsteps:

-   (1) selecting all oligopeptide of the formula AA³-AA²-AA¹ wherein    each AA independently represents an amino acid,-   (2) coupling the oligopeptide to an alkyl ester-protected    stabilizing group to form an alkyl ester-protected stabilizing    group-oligopeptide conjugate,-   (3) coupling the alkyl ester-protected-stabilizing    group-oligopeptide conjugate to a therapeutic agent by activating    the alkyl ester-protected stabilizing group-oligopeptide conjugate    with an activating agent in the presence of a therapeutic agent to    form an alkyl ester-protected stabilizing    group-oligopeptide-therapeutic agent conjugate, and-   (4) deprotecting the alkyl ester-protected stabilizing    group-oligopeptide therapeutic agent conjugate to form the compound.

A compound of the invention may also be made via the following steps:

-   (1) selecting an oligopeptide of the formula AA³-AA²-AA¹ wherein    each AA independently represents an amino acid,-   (2) coupling the oligopeptide to an allyl ester-protected    stabilizing group to form an allyl ester-protected stabilizing    group-oligopeptide conjugate,-   (3) coupling the allyl ester-protected-stabilizing    group-oligopeptide conjugate to a therapeutic agent by activating    the allyl ester-protected stabilizing group-oligopeptide conjugate    with an activating agent in the presence of a therapeutic agent to    form an allyl ester-protected stabilizing    group-oligopeptide-therapeutic agent conjugate, and-   (4) deprotecting the allyl ester-protected stabilizing    group-oligopeptide therapeutic agent conjugate to form the compound.

Yet another method for making a compound of the invention comprises thefollowing steps:

-   (1) selecting a trityl-protected oligopeptide of the formula    trityl-AA³-AA²-AA¹ wherein each AA independently represents an amino    acid,-   (2) coupling the trityl-protected oligopeptide to a therapeutic    agent by activating the trityl-protected oligopeptide with an    activating agent in the presence of a therapeutic agent, thereby    making a trityl-protected oligopeptide-therapeutic agent conjugate,-   (3) deprotecting the trityl-protected oligopeptide-therapeutic agent    conjugate under acidic conditions to form an    oligopeptide-therapeutic agent conjugate, and-   (4) coupling the oligopeptide-therapeutic agent conjugate with an    stabilizing group to form the compound.

Another possible step in connection with any of these methods isremoving uncoupled therapeutic agent by use of scavenging resin orbeads. Further, the compound may be neutralized with a pharmaceuticallyacceptable salt if desired.

Specific Compounds

Compounds of the invention include the prodrugs, Suc-Met-Ala-Leu-Dox,Gl-Met-Ala-Leu-Dox, Suc-Phe-Gly-Phe-Dnr, Suc-Phe-Gly-Leu-Dnr,Suc-Phe-Gly-Ile-Dnr, Suc-Leu-Ala-Gly-Dox, Pyg-Leu-Ala-Leu-Dnr,Suc-Leu-Ala-Leu-Dox, Suc-Leu-Thr-Leu-Dnr, Suc-Leu-Tyr-Leu-Dnr,Suc-Leu-Tyr-Leu-Dox, Suc-Met-Ala-Leu-Dnr, Suc-Met-Gly-Phe-Dnr,Suc-Met-Gly-Ile-Dnr, Suc-Met-Gly-Leu-Dnr, Suc-Tyr-Ala-Ile-Dnr, orSuc-Nle-Ala-Leu-Dnr.

Additionally, the following intermediate compounds, important to theprocess of preparation of the prodrugs of the invention, are part of theinvention:

-   Trityl-Met-Ala-Leu-Dox-   Diphenyhnethyl-Met-Ala-Leu-Dox-   Benzyloxycarbonyl-Met-Ala-Leu-Dox-   Fmoc-Leu-Met-Leu-OBn-   Met-Ala-Leu-OBn-   Methyl-succinyl-Met-Ala-Leu-OBn-   Methyl-succinyl-Met-Ala-Leu-   Fmoc-Met-Ala-Leu-   Fmoc-Met-Ala-Leu-Dnr-   Gl-Met-Ala-Leu-Dox-   Met-Ala-Leu-Dox Lactate-   Allyl-succinyl-Met-Ala-Leu-Dox-   Suc-Met-Ala-Leu-   Methyl esters of Suc-Met-Ala-Leu-   Fmoc-Met-Ala-Leu-Dox-   Methyl-succinyl-Met-Ala-Leu-Dox, and-   Allyl-hemi succinate.

EXAMPLES Example 1

Screening of Potential Prodrugs with Trouase and Human Blood

A good candidate for a prodrug with improved therapeutic index isactivated by cancer cells but relatively stable in whole human blood.Three different preparations of carcinoma were used to screen varioustest compounds. These three preparations were as follows:

-   -   (a) MCF 7/6 (breast carcinoma) cell homogenate    -   (b) MCF 7/6 (breast carcinoma) conditioned media, and    -   (c) HeLa (cervical carcinoma) cell extract anion exchange        fraction pool.

Compounds which could be hydrolyzed to a single and/or di-amino acidtoxin conjugate (i.e., AA¹-therapeutic agent and/or AA²-AA¹-therapeuticagent) were further tested for stability in whole human blood. The wholeblood was collected using commercial acid buffered citrate whole bloodcollection tubes (Becton Dickinson).

(a) Preparation of MCF 7/6 Cell Homogenate

MCF 7/6 cells were grown to confluence in a serum free medium containingDMEM:F12 (1:1), 50 mg/L bovine serum albumin, ITS-X (10 mg/L insulin,5.5 mg/L transferrin, 6.7 μg/L Na selenite, 2 mg/L ethanolamine), andLipid Concentrate (Gibco #21900-030). 100 mL of cells were harvested bycentrifugation at 4° C. 10,000×g, for 20 min and decanting thesupernatant. The pellet was resuspended in 2 mL phosphate bufferedsaline (Gibco) and centrifuged at 18,000×g for 10 min. After decantingthe supernatant, the cells (approximately 300 μL wet) were homogenizedby grinding in 1.7 mL 10 mM pH 7.2 HEPES buffer (sodium salt). Thehomogenate was centrifuged at 18,000×g at 4° C. for 5 min and thesupernatant was aliquoted and stored at ≦−20° C. for subsequent use inthe compound screen.

(b) Preparation of MCF 7/6 Conditioned Media

MCF 7/6 cells were grown to confluence in DMEM/F12 (1:1) mediumcontaining 10% fetal bovine serum, 0.05% (w/v) L-glutamine, 250 IU/mLpenicillin, and 100 μg/mL streptomycin. Cells were then washed twicewith phosphate buffered saline and incubated 24 hr at 5% CO₂, 37° C., inDMEM/F12 (1:1), 0.02% BSA, ITS-X (10 mg/L insulin, 5.5 mg/L transferrin,6.7 μg/L Na selenite, 2 mg/L ethanolamine). The conditioned media wasthen decanted and, using a stirred cell apparatus with a YM10 (10,000 MWcutoff) ultrafiltration membrane(Millipore), exchanged once with 10 mMHEPES buffer, pH 7.2 and concentrated twenty-fold. This solution wasstored in aliquots at −20° C. for use in the compound screen.

(c) Preparation of HeLa Cell Anion Exchange Fraction Pool

Thirty billion commercially produced HeLa Cells (human cervicalcarcinoma, Computer Cell Culture Ceizter, Seneffe, Belgium) werehomogenized with a sonicator and with a Dounce homogenizer in 108 mL ofaqueous lysis solution. The lysis solution contained 0.02% w/v TritonX-100, 0.04% w/v sodium azide, and a cocktail of protease inhibitors (2tablets/50 mL Complete™, EDTA-free tablets, Roche MolecularBiochemicals). The cell homogenate was centrifuged 30 minutes at 4° C.at 5000×g and the pellet was homogenized in a second 108 mL of lysissolution using a Dounce homogenizer and centrifuged as before. Thesupernatants were combined and centrifuged for 90 min at 145,000×g at 4°C.

A portion of the ultracentrifugation supernatant was diluted 2-fold witha 20 mM triethanolamine-HCl pH 7.2 buffer containing 0.01% (w/v) TritonX-100 and 0.02% (w/v) sodium azide (equilibration buffer). Thirty mL ofthe resulting solution, corresponding to approximately 180 mg ofprotein, was loaded at 4° C. on a 2.6×9.4 cm Source™ 15Q (AmershamPharmacia Biotech) low pressure anion exchange chromatography column (1ml/minute). The column was then washed with 250 ml of the equilibrationbuffer at a flow rate of 1 mL/minute. Proteins were eluted in a NaCllinear concentration gradient (0–0.5 M in the equilibration buffer,total volume of the gradient was 1000 ml) at a flow rate of 3 ml/minute.Two-minute fractions were collected and used for enzyme activitydetermination using βAla-Leu-Ala-Leu-Dox as the substrate. Itstransformation into Ala-Leu-Dox was quantified by reverse phase highperformance liquid chromatography utilizing fluorescence detection ofthe anthracycline moiety. The fractions containing the highest activitylevels were pooled (fractions #43–46; ˜0.13 M NaCl), supplemented withprotease inhibitors (Complete™, EDTA-free tablets, Roche MolecularBiochemicals), and stored as aliquots at −80° C.

(d) Cleavage Assay

Test compounds were incubated for 2 hr at 37° C. at a concentration of12.5 μg/mL, in pH 7.2, 10 mM HEPES, 1 mM CoCl₂ or 100 mM MnCl₂, with thethree different preparations of carcinoma enzyme and with whole humanblood collected over sodium citrate. Following incubation, three volumesof acetonitrile were added to stop the reaction and remove protein fromthe mixture. The sample was centrifuged at 18,000 g for 5 minutes and100 μL of supernatant was mixed with 300 μL of water prior to analysisby HPLC. For HPLC analysis 50 μL of sample was injected on a 4.6×50 mm2μ TSK Super-ODS chromatography column at 40° C. and eluted with a 3minute linear gradient from 26% to 68% acetonitrile in aqueous 20 mMammonium acetate pH 4.5 buffer at 2 mL/min. Detection was byfluorescence using an excitation wavelength of 235 nm and an emissionwavelength of 560 nm.

Test compounds that were cleaved by the trouase under the givenconditions and were stable in human blood are shown in Table 2. With fewexceptions, results for carcinoma enzyme cleavage were the same for apartially purified fraction from HeLa cells, MFC 7/6 cell homogenate,and MCF 7/6 conditioned media.

TABLE 2 Stabilizing (AA₃) (AA₂) (AA₁) Therapeutic No: Group P1 P1′ P2′Compound 1 Suc Phe Gly Phe Dnr 2 Suc Phe Gly Leu Dnr 3 Suc Phe Gly IleDnr 4 Suc Leu Ala Gly Dox 5 Pyg Leu Ala Leu Dnr 6 Suc Leu Ala Leu Dnr 7Suc Leu Ala Leu Dox 8 Suc Leu Thr Leu Dnr 9 Suc Leu Tyr Leu Dnr 10  SucLeu Tyr Leu Dox 11  Suc Met Ala Leu Dnr 12  Suc Met Ala Leu Dox 13  SucMet Gly Phe Dnr 14  Suc Met Gly Ile Dnr 15  Suc Met Gly Leu Dnr 16  SucTyr Ala Ile Dnr 17  Suc Nle Ala Leu Dnr

Example 2

Identification Tripeptide Drugs with Favorable Cleavage Rates

Test compounds were incubated for 2 hr at 37° C. at a concentration of12.5 μg/mL with HeLa cell anion exchange fraction (F1), as prepared inExample 1(c). Following incubation, three volumes of acetonitrile wereadded to stop the reaction and remove protein from the mixture. Thesample was centrifuged at 18,000 g for 5 minutes and 100 μL ofsupernatant was mixed with 300 μL of water prior to analysis by HPLC.For HPLC analysis 50 μL of sample was injected on a 4.6×50 mm 2μ TSKSuper-ODS chromatography column at 40° C. and eluted with a 3 minutelinear gradient from 26% to 68% acetonitrile in aqueous 20 mM ammoniumacetate pH 4.5 buffer at 2 mL/min. Detection was by fluorescence usingan excitation wavelength of 235 nm and an emission wavelength of 560 nm.

the amount of cleavage of each test compound was compared to the amountof a standard Suc-βAla-Leu-Ala-Leu-therapeutic agent conjugate cleavedby F1 under the same conditions. The therapeutic agent in the standard(Dox or Dnr) was the same as in the test compound. Table 3 provides therelative cleavage rates of several tripeptide prodrugs.

TABLE 3 Relative Tripeptide analog Cleavage % Suc-Nle-Gly-Phe-Dnr 17Suc-Phe-Gly-Phe-Dnr 12 Suc-Leu-Tyr-Leu-Dox* 14 Suc-Phe-Gly-Leu-Dnr 24Suc-Tyr-Ala-Ile-Dnr 25 Suc-Met-Gly-Phe-Dnr 29 Suc-Leu-Ala-Gly-Dox 40Suc-Met-Gly-Ile-Dnr 46 Suc-Met-Ala-Leu-Dox 49 Suc-Phe-Gly-Ile-Dnr 54Suc-Met-Gly-Leu-Dnr 55 Suc-Leu-Ala-Leu-Dox 62 Suc-Met-Ala-Leu-Dnr 69Pyg-Leu-Ala-Leu-Dnr 76 Suc-Leu-Ala-Leu-Dnr 76 Suc-Tyr-Ala-Len-Dnr 77*tested with recombinant rat TOP

Example 3

Tumor-Activated Prodrug Activity on LNCaP, HT-29 and PC-3 Cells

Adherent cells, LNCaP (prostate carcinoma), HT-29 (colon carcinoma) andPC-3 (prostate carcinoma), were cultured in DMEM media containing 10%heat inactivated fetal calf serum (FCS). On the day of the study thecells were detached from the plate with a trypsin solution. Thecollected cells were washed and resuspended at a concentration of0.25×10⁶ cells/ml in DMEM containing 10% FCS. 100 μl of cell suspensionwere added to 96 well plates and the plates were incubated for 3 hoursto allow the cells to adhere. Following this incubation, serialdilutions (3-fold increments) of doxorubicin or test compounds were madeand 100 μl of compounds were added per well. The plates were thenincubated for 24 hours, pulsed with 10 μl of a 100 μCi/ml ³H-thymidineand incubated for an additional 24 hours (total incubation time 48hours). The plates were harvested using a 96 well Harvester (PackardInstruments) and counted on a Packard Top Count Counter. Four parameterlogistic curves were fitted to the ³H-thymidine incorporation as afunction of drug molarity using Prism software to determine IC₅₀ values.

TABLE 4 Activity on LNCaP, HT-29 and PC-3 cells. IC50 (μM) CompoundLNCAP HT29 PC-3 DOX 0.016 0.052 0.075 Suc-Leu-Ala-Leu-Dox 1.0 36 50Suc-Ile-Ala-Leu-Dox 1.1 47 88 Suc-Leu-NMeA1a-Leu-Dox 1.2 24 45Suc-Ile-Pro-Leu-Dox 2.0 44 106 Suc-Leu-Tyr-Leu-Dox 9.4 42 51Suc-Leu-Ala-Gly-Dox 15 32 39 Suc-Leu-Tyr-Gly-Dox 15 25 64

Prostate carcinoma cells, LNCaP and PC-3 cells or colon carcinoma cellsHT-29, were incubated with increasing concentration of the indicatedcompounds for 48 hours and cellular proliferation was measured using the³H-thymidine assay. The IC₅₀ of the positive control, doxorubicin, was0.02–0.08 μM in the cell lines used. The data shows that multiple cellslines such as PC-3 and HT29 do not cleave the above-indicatedprodrugs/In contrast, an enzyme present in or on LNCAP cells cleavesseveral of the tripeptide prodrugs. The most potent analogs areexemplified with Suc-Leu-Ala-Leu-Dox and Suc-Ile-Ala-Leu-Dox, which havean IC₅₀ of approximately 1 μM on LNCaP cells.

Example 4

Suc-Leu-Ala-Leu-Dox is Better Tolerated in vivo than Doxorubicin

In a single-dose Maximum Tolerated Dose (MTD) study, groups of 5 healthyyoung female normal mice were given intravenous doses of theSuc-Leu-Ala-Leu-Dox of either 0, 23, 47, 70, 93 and 117 mg/kg,equivalent to 0, 14, 28, 42, 56, and 70 mg/kg of Doxorubicin,respectively. Suc-Leu-Ala-Leu-Dox was very well tolerated, with nomortalities observed over the 28-day study, and only slight body weightloss initially in the higher dose-groups, followed by recovery.Therefore the prodrug made it possible to safely administer anequivalent dose of doxorubicin of 66 mg/kg, which is approximately8-fold higher than would be possible with doxorubicin alone, which hasan MTD of about 4–8 mg/kg. The single-dose MTD of Suc-Leu-Ala-Leu-Doxwas not attained in this experiment, and is therefore greater than 117mg/kg.

However, post-analysis of the compounds showed that they contained about47% active compound due to water content and impurity. So the dosesmentioned in the above paragraph overestimated the amount of compoundadministered. Recalculation of the data showed that the prodrug made itpossible to safely administer an equivalent dose of doxorubicin of 70mg/kg, which is approximately 3.3-fold higher than would be possiblewith doxorubicin alone, which has an MTD of about 16 mg/kg

The above experiment was repeated and the following results wereobtained. The single dose MTD of Suc-Leu-Ala-Leu-Dox was determined tobe approximately 59 mg/kg (35 mg/kg doxorubicin equivalent), which is atleast 1.2-fold higher than that of doxorubicin alone (16 mg/kg). Themore relevant comparison is the repeat-dose (RD) MTD, as a SD is notefficacious. The repeat dose MTD of Suc-Leu-Ala-Leu-Dox wasapproximately 52 mg/kg, Q7D×5 (31 mg/kg doxorubicin equivalent, based onefficacy study in Example 12, which was 6.8-fold higher than the repeatdose MTD of doxorubicin (4 mg/kg).

Example 5

Suc-Leu-Ala-Leu-Dox is Better Tolerated In Vivo than Doxorubicin

Suc-Leu-Ala-Leu-Dox, an exemplary tripeptide prodrug of the invention,is well tolerated in mice. In a second single dose Maximum ToleratedDose (SD-MTD) study, groups of five normal ICR mice were administeredintravenous bolus doses of Suc-Leu-Ala-Leu-Dox. The mice were observeddaily for 49 days and body weights measured twice weekly. Dose levelstested were 0, 47, 59, 71, 94, 117, 140 or 164 mg/kg, equivalent to 0,28, 35, 42, 56, 70, 84 or 98 mg/kg of doxorubicin, respectively. Therewas no acute toxicity, within 24 hours, at any dose level. Dose and timedependent signs of toxicity were observed during the study. Toxicity,including partial hind-end paralysis and significant body weight loss(>20% of their initial weight), was observed in the 94 mg/kg and higherdose groups. Based on survival and lack of signs of toxicity at Day 49,the SD-MTD for Suc-Leu-Ala-Leu-Dox was determined to be 71 mg/kg(equivalent to 42 mg/kg of doxorubicin). Therefore, the SD-MTD wasapproximately 2.6-fold higher on a molar basis than the SD-MTD fordoxorubicin alone (16 mg/kg). See Table 5. This is an approximate SD-MTDdetermination based on a range of doses at 7 or 14 mg/kg doxorubicinequivalents increments over the range tested.

TABLE 5 SD-MTD SD-MTD SD-MTD Molar Ratio Compound Name (mg/kg) (mg/kgDox =) (Dox =) Doxorubicin 16 16 1   Suc-Leu-Ala-Leu-Dox 71 42 2.6

Example 6

Suc-Len-Ala-Gly-Dox is Better Tolerated In Vivo than Doxorubicin

In a second single dose Maximum Tolerated Dose (SD-MTD) study, groups offive normal ICR mice were administered intravenous bolus doses ofSuc-Leu-Ala-Gly-Dox. The mice were observed daily for 49 days and bodyweights measured twice weekly. Dose levels tested were 0, 88, 110, 132,154, 176, 198 or 220 mg/kg, equivalent to 0, 56, 70, 84, 98, 112, 126 or141 mg/kg of doxorubicin, respectively. There was no acute toxicity,within 24 hours, at any dose level. Suc-Leu-Ala-Gly-Dox was very welltolerated, with no mortalities, morbidity or significant body weightloss observed over the 49 day study at any dose level. Thus, the SD-MTDof Suc-Leu-Ala-Gly-Dox was not attained in this study, and is thereforeat least the highest dose tested. Based on survival and lack of signs oftoxicity at Day 49, the SD-MTD for Suc-Leu-Ala-Gly-Dox was at least 220mg/kg (equivalent to 141 mg/kg of doxorubicin), which is 8.8-fold higheron a molar basis than the SD-MTD for doxorubicin alone (16 mg/kg). SeeTable 6.

TABLE 6 SD-MTD SD-MTD SD-MTD Molar Ratio Compound Name (mg/kg) (mg/kgDox =) (Dox =) Doxorubicin    16    16 1   Suc-Leu-Ala-Gly-Dox >220 >1418.8

Example 7

Suc-Met-Ala-Leu-Dox is Better Tolerated In Vivo than Doxorubicin

In a single dose Maximum Tolerated Dose (MTD) study, groups of 5 healthyyoung female normal mice were given intravenous doses of theSuc-Met-Ala-Leu-Dox of either 24, 48, 71, 96 and 120 mg/kg, equivalentto 14, 28, 42, 56, and 70 mg/kg of doxorubicin, respectively. Althoughno acute signs of toxicity were observed following administration ofSuc-Met-Ala-Leu-Dox, several animals in the two highest dose groupsunderwent significant weight loss and morbidity at around 25 days postadministration. The group mean body weights of lower dose groups werenot significantly different from the vehicle-treated control group. Thesymptoms of toxicity (paralysis and morbidity) were typical ofdoxorubicin. The 28-Day survival single-dose MTD value was establishedat approximately 71 mg/kg (equivalent to about 40 mg/kg doxorubicin).The single dose MTD of Suc-Met-Ala-Leu-Dox provides a dose ofdoxorubicin about 1.5-fold higher than would be possible withdoxorubicin alone.

Example 8

Suc-Leu-Ala-Leu-Dox and its Metabolites are Rapidly Cleared

The metabolism and clearance of the prodrug compounds was studied inhealthy young female mice, administered a single intravenous dose at 117mg/kg Suc-Leu-Ala-Leu-Dox Plasma samples were obtained at 1 and 4 hours.Plasma samples of 100 μl were transferred to microcentrifuge tubes (1.5mL) and an internal standard of daunorubicin (20 μL at 0.5 mg/ml) wasadded together with acetonitrile (400 μl). The tubes were capped andbriefly vortexed followed by centrifugation at 14,000 rpm. 420 μl fromeach tube was removed and dried in vacuo. Each sample was reconstitutedin 65 μl 20 mM aqueous ammonium formate (AF) pH 4.5 buffer containingacetonitrile (20%) prior to analysis by reverse phase liquidchromatography in combination with tandem mass spectrometry (LC MS/MS).

Urine was collected at 2 and 24 hours post administration from pairs ofmice in metabolic cages. Urine samples were diluted with AF buffercontaining acetonitrile (20%) to give a target analyte concentrationwithin the practical range of the LC MS/MS assay. 30 μl of each dilutedsample was placed in a micro centrifuge tube (1.5 ml) and an internalstandard of daunorubicin (20 μL at 0.5 mg/ml) was added together with 50μl of AF buffer containing acetonitrile (20%). Each sample was thenanalyzed by LC MS/MS.

An Agilent HP1100 HPLC with DAD detector and Chemstation software wascoupled to a PE Sciex API 365 mass spectrometer with an electrospray ionsource. HPLC was performed on a TSK-Gel Super ODS, 2 mm, 4.6×50 mm(TosoHaas) reversed phase column equipped with a HAIGUARD C18 guard disc(Higgins Analytical) and stainless steel frit (Upchurch Scientific).Chromatography was performed at room temperature. The flow rate was 0.5ml/min. Injection volume was 50 μl. Gradient elution was performed usinga mobile phase of AF buffer with increasing amounts of acetonitrile. TheAPI 365 was operated at 365° C. in a multiple reaction monitoring mode,set to monitor specific analyte parent-daughter ion pairs. Integrationof chromatograms was performed by MacQuan software (PE Sciex) andquantitation of each analyte obtained by comparison to previouslyobtained calibration curves. Daunorubicin was used as an internalstandard in all cases.

The parent compound Suc-Leu-Ala-Leu-Dox was cleared from the circulationvery rapidly. After administration, 1.49%, (1 hour) and less than 0.01%(4 hours) of the administered dose could be detected in the plasma (FIG.10). At 2 and 24 hours, the urine contained 9.31 and 2.11% of theadministered dose, respectively (FIG. 11). Low levels of the majorpeptide metabolites, Ala-Leu-Dox and Leu-Dox, as well as Dox could bedetected in plasma. Leu-Dox in particular could be detected in the urineand, as with the other metabolites, was higher at both 2 hours and 24hours than in plasma at 1 hour or 4 hours. Little free doxorubicin waspresent in blood or urine at any time point.

TABLE 7 Plasma* Urine* 1 hr 4 hr 2 hr 24 hr Suc-Leu-Ala-Leu-Dox 1.49 0.011 9.31 2.11 Ala-Leu-Dox 0.006 0.000 0.02 0.01 Leu-Dox 0.019 0.0011.53 0.45 Dox 0.002 0.000 0.05 0.15 *Percent of administered dose

The levels of the prodrug and its metabolites detected in the urine showthat the kidney is a major organ of excretion. In contrast, doxorubicinis known to be cleared principally through the hepato-biliary system.The clearance and metabolism profiles of Suc-Leu-Ala-Leu-Dox suggestthat there is a very small amount of cleavage and activation of theprodrug in the circulation, or tissues of normal mice. The prodrug andmetabolites appear rapidly in the urine, with Leu-Dox being the majormetabolite species detected in urine, as well as plasma. In normalanimals, clearance of the inactive Suc-Leu-Ala-Leu-Dox and metaboliteforms appears to predominate over cleavage to the toxic doxorubicin. Theclearance/metabolism results are consistent with the good safety profileobserved in the toxicity studies, probably because in normal mice theprodrug, and low levels of metabolites formed, are rapidly cleared fromthe circulation and excreted via the urine. Thus very little of theprodrug remains available in the circulation to release freedoxorubicin, which would be toxic to normal tissues.

Example 9

Suc-Met-Ala-Leu-Dox and its Metabolites are Rapidly Cleared

The metabolism and clearance of the prodrug compounds was studied inhealthy young female mice, administered a single intravenous dose at 120mg/kg Suc-Met-Ala-Leu-Dox. Plasma samples were obtained at 1 or 4 hours,and urine was collected at 2 and 24 hours post administration. Levels ofthe prodrug and metabolites were measured by LC-MS with UV detection ofdoxorubicin.

Suc-Met-Ala-Leu-Dox was detected in plasma at very low levels, with onlyabout 0.1% and 0.001% of the administered dose detected at 1 and 4 hoursrespectively (FIG. 12). At 2 and 24 hours the urine contained 6.6% and1.4% of the administered dose of prodrug (FIG. 13). Low levels of themajor peptide metabolites, Ala-Leu-Dox and Leu-Dox, as well as Dox couldbe detected in plasma. In plasma, Leu-Dox levels were about 10 timeshigher than Ala-Leu-Dox at both 1 and 4 hours and accumulatedsignificantly in the urine by 2 hours. These results suggest that therewas significant cleavage of the methionine-containing prodrug. Littlefree doxorubicin was present in plasma, while urine levels were higherat 2 and 24 hours, suggesting that the rapid disappearance of theprodrug from the plasma may be due in part to cleavage to the activemetabolite, doxorubicin.

TABLE 8 Plasma Urine 1 hr 4 hr 2 hr 24 hr Suc-Met-Ala-Leu-Dox 0.1020.001 6.64 1.44 Ala-Leu-Dox 0.002 0.000 0.16 0.02 Leu-Dox 0.022 0.0017.28 1.25 Dox 0.002 0.002 0.30 0.61 Percent of administered dose

The levels of the prodrug and its metabolites detected in the urine showthat the kidney is a major organ of excretion. In contrast, doxorubicinis known to be cleared principally through the hepato-biliary system.The clearance and metabolism studies with Suc-Met-Ala-Leu-Dox suggestthat there was considerable systemic or tissue cleavage and activationof the prodrug in normal mice possibly in kidney based on the high urinemetabolite values, relative to plasma. Because Leu-Dox is known to betaken up by cells, and cleaved to the active toxic compound doxorubicin,the significant levels of Leu-Dox and doxorubicin are consistent withthe less well tolerated safety profile of Suc-Met-Ala-Leu-Dox. Thesteady levels of doxorubicin in plasma and urine in normal mice are alsoconsistent with slow kinetics of this compound.

Example 10

Comparative Metabolism in Mice

Four groups of ICR normal female mice were administered a single IVbolus dose with approximately 100 μmol/Kg of Suc-βAla-Leu-Ala-Leu-Doxand compared to Suc-Leu-Ala-Leu-Dox, Suc-Leu-Ala-Gly-Dox or 10 μl mol/Kgof doxorubicin (Dox). Plasma was obtained from three individual animalsin each group at 5 minutes, 1, 2, 4, or 6 hr. Parent,dipeptidyl-doxorubicin (AL-Dox, AG-Dox), α-aminoacyl-doxorubicin (L-Doxor G-Dox) and doxorubicin concentrations were analyzed in extracts ofthe plasma samples using a reverse phase gradient HPLC method withfluorescence detection (λex=480 nm, λem=560). Peak retention time ifG-Dox was not confirmed because a standard was not available. Quantitieswere determined using a linear standard curve fit to measurements of 10to 2000 ng/mL doxorubicin solutions in mouse plasma.

Concentration time courses indicate that metabolic patterns were similarfor all compounds with the exception of Suc-Leu-Ala-Gly-Dox. Inparticular, except for Suc-Leu-Ala-Gly-Dox, L-Dox was the majormetabolite over the first two hr while the dipeptidyl-conjugate AL-Doxwas a more minor product that formed at about the same time as L-Dox.Doxorubicin appeared later with the plasma concentration decreasing moreslowly over time than the other metabolites as expected from the currentand previously measured doxorubicin pharmacokinetic profiles(Tabrizi-Fard et al., “Evaluation of the Pharmacokinetic Properties of aDoxorubicin Prodrug in Female ICR (CD1® Mice following intravenousadministration,” Proc American Association for Cancer Research, 42: 324(2001)) and by the doxorubicin control group. The observed cleavagepattern is initial activation of the tripeptide prodrug by anendopeptidase acting between P2-P1, or between the P1 and P1′ aminoacids. Doxorubicin increases after exopeptidase cleavage of unprotectedpeptide-doxorubicin metabolic intermediates of the prodrug. Areas underthe plasma concentration time curves indicate dosing withSuc-Leu-Ala-Gly-Dox the only peptidyl-conjugate not predicted to becleaved by CD10, resulted in considerably (ten times) less systemicdoxorubicin exposure in normal animals than the two CD10 cleavabletripeptidyl-doxorubicin compounds, or Suc-βAla-Leu-Ala-Leu-Dox. The TOPcleavable tripeptide (Suc-Leu-Ala-Leu-Dox) produced about half thedoxorubicin exposure (AUC) as compared to that observed with equimolardoses of the tetrapeptide (Suc-βAla-Leu-Ala-Leu-Dox). It should be notedthat relative Doxorubicin exposure after dosing these compounds TABLE 9is consistent with the relative safety expressed as maximum tolerateddose in a mouse safety study, wherein Suc-Leu-Ala-Gly-Dox wassignificantly less toxic, when tested up to 3× the molar equivalentdose. Suc-Leu-Ala-Leu-Dox was better tolerated thanSuc-βAla-Leu-Ala-Leu-Dox and doxorubicin. Thus, the tripeptide prodrugssignificantly protect mice from systemic exposure to doxorubicin afterplasma cleavage following administration of 10 times higher molarequivalent doses. The tripeptides, especially Suc-Leu-Ala-Gly, resultedin lower systemic exposures. Thus, the tripeptide, especiallySuc-Leu-Ala-Gly, conveys more protection from unwanted systemictoxicities.

TABLE 9 AL-Dox L-Dox or AG- or G- Parent DOX Dox Dox Dosed Compound (μM· hr) (μM · hr) (μM · hr) Suc-βAla-Leu-Ala-Leu-Dox 806 3.2 40   3.4Suc-βAla-Ile-Ala-Leu-Dox 326 0.5 8.5 1.5 Suc-Leu-Ala-Leu-Dox 634 1.6 5.11.6 Suc-Ile-Ala-Leu-Dox 452 1.0 6.2 0.9 Suc-Leu-Ala-Gly-Dox 310 2.1 0.90.3 Doxorubicin (Dox) N/A N/A N/A 3.3

Example 11

Suc-Leu-Ala-Leu-Dox is Well Tolerated in Tumor Bearing Mice

The Suc-Leu-Ala-Leu-Dox prodrug was also safe under repeat-doseconditions, when administered intravenously every 5 days for five dosesat either 52 or 63 mg/kg, equivalent to 31 and 38 mg/kg of doxorubicinto tumor-bearing nude mice. In two separate studies with groups of 8 or10 mice each, no acute signs of toxicity were observed following repeatadministration of Suc-Leu-Ala-Leu-Dox. In one study, one animal in eachdose group (n=8) was terminated as a possible drug-related toxicitydeath late in the study, at Days 58 and 57 in the low and highdose-groups respectively. The safety of the compound was evidenced bylittle body weight loss in either the high or low dose groups (less than5% of the initial weight) during the 60-day observation period, insurviving animals (FIG. 14). In the other study, both the high and lowdose were well tolerated. Only one animal in the high dose group (n=10)had over 15% of body weight loss at the end of the study (Day 61).

Example 12

Suc-Leu-Ala-Leu-Dox is Effective in Tumor Xenograft Models

Two mouse xenograft studies demonstrated the efficacy ofSuc-Leu-Ala-Leu-Dox on the growth of human colon carcinoma (LS174T), andthe outcome in terms of long-term survival. Healthy young female nudemice were subcutaneously implanted with chunks of LS174T. When thetumors reached approximately 50–100 mg in weight, treatment every 5 daysfor 5 doses, of groups of 8 or 10 mice with either 0, 52 or 63 mg/kgSuc-Leu-Ala-Leu-Dox was initiated. Tumor size and body weights weremeasured twice weekly for up to 60 days.

A dose-dependent increase in survival of the mice was observed. The MeanDay of Survival was increased to 20 and 32 days in the treated groups,compared with 18 days in the vehicle control group (FIG. 15).Suc-Leu-Ala-Leu-Dox decreased the rate of tumor growth considerably overthe vehicle control group (FIG. 16). In a greater number of cases thanthe untreated control, the tumors of the treated animals appeared tostop growing or regress. The compound was well tolerated, suggestingthat the administered doses were both below the repeat-dose MTD.

In tumor efficacy studies, Suc-Leu-Ala-Leu-Dox exhibited dose-dependentefficacy, delaying tumor growth and increasing survival, and was verywell tolerated. It was significantly more efficacious in thisdoxorubicin-insensitive xenograft model than doxorubicin alone. Despiterapid excretion of the parent compound, Suc-Leu-Ala-Leu-Dox, intumor-bearing mice it has surprisingly strong anti-tumor activity,delivering high dose-levels of doxorubicin while sparing systemictoxicity. Thus it is an effective prodrug, which is able to extendsurvival and delay tumor growth in a doxorubicin-insensitive model, inwhich doxorubicin alone is virtually inactive.

However, post-analysis of the compounds showed that they contained about47% active compound due to water content and impurity. So the dosesmentioned in the above paragraph overestimated the amount of compoundadministered. Recalculation of the data showed that Suc-Leu-Ala-Leu-Doxdecreased the rate of tumor growth over the vehicle control group. Intumor efficacy studies, Suc-Leu-Ala-Leu-Dox exhibited dose-dependentefficacy, delaying tumor growth and increasing survival, and was welltolerated. It was more efficacious in this doxorubicin-insensitivexenograft model than doxorubicin alone.

Example 13

Suc-Leu-Ala-Gly-Dox is Effective in Tumor Xenograft Models

A mouse xenograft study demonstrated the efficacy of Suc-Leu-Ala-Gly-Doxon the growth of human colon carcinoma (HT-29), and the outcome in termsof long-term survival. Healthy young male nude mice were subcutaneouslyinjected with 5 million of HT-29 cells. When the tumors reachedapproximately 100 mg in weight, treatment every 7 days for 5 doses, ofgroup of 8, 10, or 12 mice with vehicle, 4 mg/kg doxorubicin, 40 or 67mg/kg Suc-Leu-Ala-Gly-Dox was initiated. Tumor size and body weightswere measured twice weekly for up to 60 days.

A dose-dependent increase in survival of the mice was observed.Suc-Leu-Ala-Gly-Dox, given at 40 mg/kg and 67 mg/kg (42 mg/kg and 70mg/kg doxorubicin equivalent concentration) was very well tolerated andgreatly prolonged survival of tumor bearing mice. The doxorubicintreated mice had a MDS of 30 days. The Mean Day of Survival wasincreased to 39 and 41 days in the treated groups withSuc-Leu-Ala-Gly-Dox, compared with 26 days in the vehicle control group.(TABLE 10). The high does of Suc-Leu-Ala-Gly-Dox decreased the rate oftumor growth significantly over the vehicle control group (TABLE 10).The compound was very well tolerated, suggesting that the administereddoses were both below the repeat-dose MTD.

TABLE 10 Calculated Median Mean Day Extension of Toxicity Mean Tumor %TGI Tumor % TGI of Mean Day of Number of (Weight Dose Weight at Day 14(Day 14, Weight at Day (Day 25, Survival Survival over Long Term Loss >Compound (mg/kg) (mg) mean) 25 (mg) median) (day) controls Survivors20%) saline — 733 ± 72    0% 1515   0% 25.7 ± 2.6   0%  0/11  0/11  (n =12)  (n = 11) Doxorubicin   4.0 665 ± 103 9.3% 1046 30.9% 30.0 ± 3.716.7%  0/10  1/10  (n = 10) (n = 9) Suc-Leu-Ala- 40 704 ± 92  4.0%  95237.2% 38.8 ± 6.6 51.0% 1/7 0/7 Gly-Dox (n = 8) (n = 7) Suc-Leu-Ala- 67490 ± 124 33.2%*  857 43.5% 41.3 ± 7.2  60.7%* 3/7 0/7 Gly-Dox (n = 7)(n = 7) *Statistically different from the control at the p level of 0.10(two-tailed) #: Some mice were excluded from tumor growth/survivalanalyses due to ulceration of tumors. TGI: Tumor Growth Inhibition overcontrol

Example 14

Advantages of Prodrugs over the Unconjugated Therapeutic Agent

The prodrugs of the invention provide treatment advantages over thetherapeutic agent in its unconjugated form.

In the single dose Maximum Tolerated Dose (SD-MTD) studies, groups ofnormal mice were administered intravenous bolus doses of the prodrug.The mice were observed daily for 60 days and body weights measured twiceweekly. The SD-MTD was estimated to be equal to the highest dose thatproduced no death in mice after 60 days. As shown in Table 11, thesingle-dose MTD of the prodrugs range from 10-fold to at least 16-foldhigher than that of doxorubicin alone.

TABLE 11 Optimal Optimal Efficacy Efficacy Relative SD MTD* Repeat DoseDose Rate of Compound (mg/kg) (mg/kg) Frequency Cleavage Suc-Met-Ala- 70(42) n.d. n.d. 49 Leu-Dox Suc-Leu-Tyr- >128 (70)    n.d. n.d. 14 Leu-DoxSuc-Leu-Ala- >117 (70)    63 (38) Q 5 days × 5 62 Leu-Dox Suc-βAla-Leu-75 (42) 71.2 (40)   Q 1 week × 5 100  Ala-Leu-Dox Doxorubicin 4 (4) 4(4) Q 1 week × 5 n.a. n.d. = not determined; n.a. = not applicable

In repeat-dose studies in tumor bearing mice, groups of ten mice weredosed with various amounts of prodrug for a total of five doses ateither five day or 1 week intervals. After frequent observation over 60days, the dose which proved to be within acceptable toxicity limits andproduced a decrease in tumor size was identified as the optimal efficacyrepeat dose. As seen in Table 11, optimal efficacy repeat dose of theprodrugs are approximately 9-fold higher than that of doxorubicin alone.Thus, the prodrugs permit a much greater amount of therapeutic agent tobe delivered to the body as a whole and thus to the vicinity of thetarget cell.

Surprisingly, the tripeptide prodrug, Suc-Leu-Ala-Leu-Dox, which iscleaved less completely than Suc-βAla-Leu-Ala-Leu-Dox in in vitro enzymeassays (see, e.g., Examples 1 and 2), is significantly better toleratedand more efficacious when administered in vivo at an equivalent dose.Under these conditions, Suc-Leu-Ala-Leu-Dox was well tolerated, andbelow its MTD threshold, when given repeatedly on a 5-day dosingschedule, compared with a 7-day dosing schedule forSuc-βAla-Leu-Ala-Leu-Dox, which was close to its MTD. The lower toxicityof Suc-Leu-Ala-Leu-Dox is consistent with the results of the metabolismstudies. The predominant plasma metabolites of Suc-Ala-Leu-Dox areAla-Leu-Dox and Leu-Dox. This is the same metabolite pattern asSuc-βAla-Leu-Ala-Leu-Dox, which is cleaved by trouase. The relativeamounts of the metabolites detected in plasma, however, are less withthe tripeptide.

A similar pattern of an upward shift in MTD value was observed withanother tripeptide, Suc-Leu-Tyr-Leu-Dox, which was also well-toleratedat relatively high doses and was poorly cleaved by trouase.

The substantially higher MTD value of Suc-Leu-Ala-Leu-Dox is consistentwith the in vitro evidence that tripeptides are not as good substratesfor enzymatic cleavage as compared with the tetrapeptide,Suc-βAla-Leu-Ala-Leu-Dox. The resultant slower cleavage rates yieldlower levels of systemic doxorubicin and hence less toxicity. Theunexpectedly good efficacy results for Suc-Leu-Ala-Leu-Dox indicate thatin the vicinity of a tumor, cleavage of tripeptides appears to be moreeffective than the background level of systemic cleavage. Based on thegood therapeutic window observed with Suc-Leu-Ala-Leu-Dox, it appearsthat there is marked selectivity of the tripeptides for tumor tissueover the rest of the body. Considering this and the improved therapeuticwindow of the Suc-Leu-Ala-Leu-Dox compared with doxorubicin, is can beextrapolated that the Suc-Leu-Ala-Leu-Dox should be particularly usefulfor clinical treatment of tumors.

However, post-analysis of the compounds showed that they contained about47% active compound due to water content and impurity. So the dosesmentioned in the above paragraphs overestimated the amount of compoundadministered. The results from the experiment described above wasre-analyzed. The re-analyzed data and new data for Suc-Leu-Ala-Gly-Doxis presented in TABLE 12.

TABLE 12 Repeat Dose Repeat Relative SD MTD MTD Dose Rate of Compound(mg/kg)* (mg/kg)* Frequency Cleavage Suc-Mer-Ala- 70 (42) n.d. n.d. 49Leu-Dox Suc-Leu-Tyr- >128 (70)    n.d. n.d. 14 Leu-Dox (56)Suc-βAla-Leu- 50 (28) 57 (32) Q 7 D × 5 100  Ala-Leu-Dox Suc-Leu-Ala- 59(35) 52 (31) Q 5 D × 5 62 Leu-Dox Suc-Leu-Ala- >220 (141)   >110 (70)   Q 7 D × 5 40 Gly-Dox Doxorubicin 16 (16) 4 (4) Q 7 D × 5 n.a. n.d. = notdetermined; n.a. = not applicable *values in parentheses are thedoxorubicin equivalent dose

Recalculation of the data showed that the single-dose MTD of theprodrugs range from 0.8-fold to at least 7.8-fold higher than that ofdoxorubicin alone. After frequent observation over 60 days, the dosewhich proved to be within acceptable toxicity limits was identified asthe maximum tolerated repeat dose. As seen in TABLE 12, RD-MTD of theprodrugs are approximately 6.8-fold higher than that of doxorubicinalone. Repeat dosing of the prodrugs at or lower than their RD-MTDsignificantly prolong survival of LS174t or HT-29 tumor bearing mice,whereas that of doxorubicin is ineffective. As seen in TABLE 12, optimalefficacy repeat dose of the prodrugs are approximately 6-fold higherthan that of doxorubicin alone.

A similar pattern of an upward shift in MTD value was observed withtripeptides, Suc-Leu-Ala-Gly-Dox and Suc-Leu-Tyr-Leu-Dox, which werealso well tolerated at relatively high doses and was poorly cleaved bytrouase.

The higher MTD value of Suc-Leu-Ala-Leu-Dox and Suc-Leu-Ala-Gly-Dox areconsistent with the in vitro evidence that tripeptides are not as goodsubstrates for enzymatic cleavage as compared with the tetrapeptide,Suc-βAla-Leu-Ala-Leu-Dox. The resultant slower cleavage rates yieldlower levels of systemic doxorubicin and hence less toxicity. Theunexpectedly good efficacy results for Suc-Leu-Ala-Leu-Dox andSuc-Leu-Ala-Gly-Dox indicate that in the vicinity of a tumor, cleavageof tripeptides appears to be more effective than the background level ofsystemic cleavage. Based on the good therapeutic window observed withSuc-Leu-Ala-Leu-Dox and Suc-Leu-Ala-Gly-Dox, it appears that there ismarked selectivity of the tripeptides for tumor tissue over the rest ofthe body. Considering this and the improved therapeutic window of theSuc-Leu-Ala-Leu-Dox and Suc-Leu-Ala-Gly-Dox compared with doxorubicin,is can be extrapolated that the Suc-Leu-Ala-Leu-Dox andSuc-Leu-Ala-Gly-Dox should be particularly useful for clinical treatmentof tumors.

Example 15

Hydrolysis by Purified CD10

Equal amounts of purified Porcine Kidney CD10 (Elastin Products Company)were incubated with 12.5 μg/mL of various peptidyl doxorubicin compoundsfor up to 10 hr at 37° C. in pH 7.4 50 mM TrisHCl, 150 mM NaCl, 0.1%Triton X-100. Reaction products were analyzed by HPLC with fluorescencedetection. Rates were essentially linear over the incubation period. Theobserved product was Leu-doxorubicin. Table 13 provides the percent ofeach test compound that was hydrolyzed over the ten hour period. Furtherthese results are expressed relative to a standard test compound,Suc-βAla-Leu-Ala-Leu-Dox.

TABLE 13 Hydrolysis by CD10 Fraction hydrolyzed Substrate %hydrolysis/10 hr relative to standard Suc-βAla-Leu-Ala-Leu-Dox 10.9 1.0(Standard) Suc-Leu-Ala-Leu-Dox 8.3 0.75 Suc-Leu-Ala-Gly-Dox 0 0

Example 16

Tumor-Activated Prodrug Activity on Ball-1, Ramos and Namalwa Cells

In addition to LNCaP, a number of B-cell lines, such as Ramos andNamalwa cells express CD10 (CD10^(pos) cells). However, another B-cellline, BALL-1 cells, do not express CD10 and serve as a CD10 negativecell line (CD10^(neg) cells).

Suspension cells, BALL-1, Ramos and Namalwa cells were cultured in RPMImedia containing 10% heat inactivated fetal calf serum (FCS). On the dayof the study, the cells were collected, washed and resuspended at aconcentration of 0.5×10⁶ cells/ml in RPMI containing 10% FCS. 100 μl ofcell suspension was added to 96 well plates. Serial dilutions (3-foldincrements) of doxorubicin or test compounds were made and 100 μl ofcompounds were added per well. Finally 10 μl of a 100 μCi/ml³H-thymidine was added per well and the plates were incubated for 24hours. The plates were harvested using a 96 well Harvester (PackardInstruments) and counted on a Packard Top Count counter. Four parameterlogistic curves were fitted to the ³H-thymidine incorporation as afunction of drug molarity using Prism software to determine IC₅₀ values.

The most selective analog was Suc-Leu-Ala-Leu-Dox (Table 14) with an˜30-35 fold difference between CD10^(pos) and CD10^(neg) cells.

TABLE 14 Activity on BALL (CD10^(neg))-1, Ramos (CD10^(pos)) & Namalwa(CD10^(pos)) cells IC50 (μM) Ratio Ball- Ball-1: Ball-1: Compound 1Ramos Namalwa Ramos Namalwa DOX  0.02 0.01 0.01 1 2 Suc-Leu-Ala-Leu-Dox78.67 2.63 2.27 30  35  Suc-Leu-Ala-Gly-Dox 23.33 22.67  17.33  1 1Thus, Suc-Leu-Ala-Leu-Dox is cleaved by CD10 but Suc-Leu-Ala-Gly-Dox isnot.Analytical Methods for the Remaining Examples

The peptide sequences, synthesized using either solid or solution phaseapproaches, were used without further purification if the analyticalHPLC (methods A, B & D) showed the crude product to be greater than 80%pure. If not, the material was purified using preparative HPLC Method C.

HPLC Method A

Analytical HPLC analyses were performed on a Waters 2690 using a C-18column (4 μm, 3.9×150 mm ID, flow rate 1 mL/min) eluting with a gradientof solvent A (0.1% TFA/H₂O) and solvent B (0.1% TFA/ACN) and the datawas processed at λ254 nm using the Waters Millennium system. AnalyticalHPLC gradient started with 90% of solvent A and ended with 100% ofsolvent B over a period of 14 minutes (linear). Purity of the compoundsfor this method and the following ones was assessed as the relativepercentage area under the curve of the peaks.

HPLC Method B

Analytical HPLC analyses were performed on a Waters 2690 using a C-8column (3.5 μm, 4.6×150 mm ID, flow rate 1 mL/min) eluting with agradient of solvent A (80% 20 mM ammonium formate and 20% acetonitrile)and solvent B (20% 20 mM ammonium formate and 80% acetonitrile) and thedata was processed at λ254 nm using the Waters Millennium system.Analytical HPLC gradient started with 100% of solvent A to 100% ofsolvent B over a period of 30 minutes (linear).

HPLC Method C

Preparative purification of crude products was achieved using a WatersDelta Prep 4000 system using a C-4 column (15 μm, 40×100 mm ID, flowrate 30 mL/min) eluting with a gradient of solvent A (H₂O), and solventB (MeOH). The preparatory HPLC gradient started with 80% of solvent Aand goes to 100% of solvent B over a period of 70 minutes (linear). Thedata was processed at λ254 nm using the Waters Millennium System.

HPLC Method D

Analytical HPLC was accomplished on a Hewlett Packard instrument using aTSK superODS column (TosoHaas); solvent A (TFA 0.1% in water); solvent B(TFA 0.1% in acetonitrile); gradient: 30 to 36% of B in 2 minutes, 36 to41% of B in 10 minutes, 41 to 90% of B in 3 minutes, 5 minutes at 90% B,detection wavelength λ254 nm.

NMR and MS

Additional structural determinations were done by NMR and MS techniquesand the results supported the claimed compounds.

TLC Method

TLC analysis was carried out on silica gel 60F-254 nm-0.25 mm plates(Merck) with DCM/MeOH/H₂O/Formic acid 88% 85/15/1/2 for elution.

Ninhydrin Test

A few milligrams of product were introduced in a test tube, and twodrops of Solution A (50 mg/mL ninhydrin in ethanol), two drops ofSolution B (4 mg/mL phenol in ethanol), then two drops of Solution C (2mL 0.01M KSCN, aqueous in 100 mL pyridine) were added. The mixture wasleft in a boiling water bath for five minutes. In the presence of a freeamine the solution becomes purple.

Specific Oligopeptide Synthetic Examples

Sources of Commercially Available Reagents

Doxorubicin and Daunorubicin were supplied by Meiji (Japan), Pd(PPh₃)₄by Strem chem (Newburyport, Mass.), PEG by Shearwater(Huntsville, Ala.),solvents, HATU by Aldrich (Milwaukee, Wis.); all resins and amino acidswere supplied by ABI (Foster City, Calif.), Novabiochem (San Diego,Calif.), Advanced ChemTech (Louisville, Ky.), Peptide International(Louisville, Ky.), or SynPep (Dublin, Calif.).

Example 17

Synthesis of Fmoc-Leu-Ala-Leu-OH

Tripeptide (Fmoc-Leu-Ala-Leu-OH) was synthesized using solid-phaseapproach with standard Emoc chemistry. A typical synthesis used Wang'salkoxy resin (0.60 mmol/gm loading). Fmoc-protected amino acids wereused for solid-phase peptide synthesis.

For a scale of 1 mM peptide on resin, 3 equivalents of amino acid waspreactivated with HBTU as the activating agent for 5 minutes beforebeing added to the resin together with 2 equivalents of DIEA. Thecoupling reaction was carried out for 2 h and then washed with DMF (25mL×3) and DCM (25 mL×3). The coupling reaction was repeated using 2equivalents of amino acid using similar conditions. The reactionprogress was monitored using ninhydrin test and if the ninhydrin testindicated incomplete reaction after 2 h then the coupling step wasrepeated for a third time. Deprotection was accomplished using 20%piperidine in DMF for 15–20 minutes. The coupling step was repeated withthe next amino acid until the desired peptide was assembled on resin.The final cleavage of peptide from the resin was accomplished bytreating the resin with a solution of 95% TFA and 5% water. Afterstirring the reaction mixture for 2 h at rt, the resin was filteredunder reduced pressure and washed twice with TFA. Filtrates werecombined and the peptide was precipitated by adding 400 mL of coldether. The peptide was filtered under reduced pressure and dried toyield Fmoc-Leu-Ala-Leu-OH (92% HPLC purity by method A). Crude peptidewas characterized by LC/MS and used for the next step without anyfurther purification.

Example 18

Synthesis of Fmoc-Met-Ala-Leu-OH

Tripeptide (Fmoc-Met-Ala-Leu-OH) was synthesized using solid-phaseapproach with standard Fmoc chemistry. A typical synthesis used Wang'salkoxy resin (0.60 mmol/gm loading). Fmoc-protected amino acids wereused for solid-phase peptide synthesis. For a scale of 1 mM peptide onresin, 3 equivalents of amino acid were preactivated with HBTU as theactivating agent for 5 minutes before being added to the resin togetherwith 2 equivalents of DIEA. The coupling reaction was carried out for 2h and then washed with DMF (25 mL×3) and DCM (25 mL×3). The couplingreaction was repeated using 2 equivalents of amino acid under similarconditions. The reaction progress was monitored using ninhydrin test andif the ninhydrin test indicated incomplete reaction after 2 h then thecoupling step was repeated for a third time. Deprotection wasaccomplished using 20% piperidine in DMF for 15–20 minutes. The couplingstep was repeated with the next amino acid until the desired peptide wasassembled on resin. The final cleavage of peptide from the resin wasaccomplished by treating the resin with a solution of 95% TFA and 5%water. After stirring the reaction mixture for 2 h at rt, the resin-wasfiltered under reduced pressure and washed twice with TFA. Filtrateswere combined and the peptide was precipitated by adding 400 mL of coldether. The peptide was filtered under reduced pressure and dried toyield Fmoc-Met-Ala-Leu-OH (90% HPLC purity by method A). Crude peptidewas characterized by LC/MS and used for the next step without anyfurther purification.

Example 19

Synthesis of Fmoc-Leu-Ala-Leu-Dox

Doxorubicin.HCl (2.34 g, 4.03 mmol) and Fmoc-Leu-Ala-Leu-OH (2.4 g, 4.48mmol) were dissolved at room temperature in anhydrous DMF (150 mL). Tothis rapidly stirred solution, DIEA (1.56 mL, 8.96 mmol) was added inone portion and the reaction mixture was stirred for 15 minutes at roomtemperature. The reaction mixture was cooled to 0° C. using an ice bathand 1.87 g (4.92 mmol) of HATU was added slowly over 10 minutes. Thereaction mixture was stirred for another 60 minutes at room temperature.Ice cold water (200 mL) was added to the reaction mixture, whichresulted in the formation of a red precipitate. The precipitate wascollected over a coarse frit, washed with 3×50 mL water and 3×50 diethylether and dried under reduced pressure to yield Fmoc-Leu-Ala-Leu-Dox(89% yield, 94% HPLC purity by method A). This product was characterizedby MS and used for the next step without any further purification.

Example 20

Synthesis of Fmoc-Met-Ala-Leu-Dox

Doxorubicin.HCl (2.26 g, 3.89 mmol) and Fmoc-Met-Ala-Leu-OH (2.4 g, 4.33mmol) were dissolved at room temperature in anhydrous DMF (150 mL). Tothis rapidly stirred solution, DIEA (1.5 mL, 8.66 mmol) was added in oneportion and the reaction mixture was stirred for 15 minutes at roomtemperature. The reaction mixture was cooled to 0° C. using an ice bathand 1 g (2.64 mmol) of HATU was added slowly over 10 minutes. Thereaction mixture was stirred for another 60 minutes at room temperature.Ice cold water (200 mL) was added to the reaction mixture, whichresulted in the formation of a red precipitate. The precipitate wascollected over a coarse frit, washed with 3×50 mL water and 3×50 diethylether and dried under reduced pressure to yield Fmoc-Met-Ala-Leu-Dox(86% yield, 93% HPLC purity by method A). This product was characterizedby MS and used for the next step without any further purification.

Example 21

Synthesis of Suc-Leu-Ala-Leu-Dox

To a solution of Fmoc-Leu-Ala-Leu-Dox (4.4 g, 4.13 mmol) in 20 mL of dryDMF, piperidine (20.4 mL, 206 mmol) was added in one portion resultingin a color change from red to purple. The reaction mixture was stirredfor 5 minutes at room temperature and then cooled to −20° C. using dryice/acetone bath. 21.2 g (210 mmol) of succinic anhydride was then addedto the cooled reaction mixture in one portion. The reaction was stirredrapidly at −5° C. for 5 minutes then at room temperature for another 90minutes. 750 mL of anhydrous diethyl ether was added to the reactionmixture, which resulted in the formation of a red precipitate. Thisprecipitate was isolated on a medium glass frit, washed with 2×50 mL ofdiethyl ether and dried under reduced pressure to yieldSuc-Leu-Ala-Leu-Dox (80% yield, 88% HPLC purity by method B). The finalproduct was purified using prep HPLC method C and characterized by LC/MSwhich gave a molecular weight of 939 (expected molecular weight 940).

Example 22

Synthesis of Suc-Met-Ala-Leu-Dox

To a solution of Fmoc-Met-Ala-Leu-Dox (4 g, 3.7 mmol) in 50 mL of dryDMF, piperidine (18.72 mL, 185 mmol) was added in one portion resultingin a color change from red to purple. The reaction mixture was stirredfor 5 minutes at room temperature and then cooled to −20° C. using dryice/acetone bath. 19 g (189 mmol) of succinic anhydride was then addedto the cooled reaction mixture in one portion. The reaction was stirredrapidly at −5° C. for 5 minutes then at room temperature for another 90minutes. 750 mL of anhydrous diethyl ether was added to the reactionmixture, which resulted in the formation of a red precipitate. Thisprecipitate was isolated on a medium glass frit, washed with 2×50 mL ofdiethyl ether and dried under reduced pressure to yieldSuc-Met-Ala-Leu-Dox (78% yield, 89% HPLC purity by method B). The finalproduct was purified using prep HPLC method C and characterized by LC/MSwhich gave a molecular weight of 939 (expected molecular weight 940).

Example 23

Synthesis of Fmoc-Met-Ala-Leu-OBn

The Fmoc-Met-Ala-Leu is added into a round bottom flask with DMF and amagnetic stirrer. After the tripeptide is dissolved, benzyl bromide,followed by cesium carbonate, is added to the solution with stirring.The reaction mixture is stirred at room temperature for 1.5 hrs. Then,the reaction mixture is slowly poured into a flask with iced water. Theprecipitate is collected by suction filtration. The product,Fmoc-Met-Ala-Leu-OBn, is washed with water and placed in a vacuumdesiccator.

Example 24

Synthesis of Met-Ala-Leu-OBn

In a round bottom flask (25 mL), Fmoc-Met-Ala-Leu-OBn is dissolved in 5mL of anhydrous DMF. Piperidine is added to the solution and the mixtureis stirred at room temperature for 25 minutes. The reaction is quenchedwith water and extracted with ethyl acetate. The combined organic layeris further washed by water, brine and dried over sodium sulfate. SolidMet-Ala-Leu-OBn is obtained after removal of solvent.

Example 25

Synthesis of MeOSuc-Met-Ala-Leu-OBn

In a round bottom flask, methyl hemisuccinate is dissolved in anhydrousDMF. DIEA followed by HBTU are added into the solution. The mixture isstirred at room temperature for 45 minutes. To this mixture is added asolution of Met-Ala-Leu-OBn (crude) in anhydrous DMF. The mixture iscontinually stirred at room temperature for 2.5 hrs. Then, the reactionmixture is slowly poured into a flask with iced water while stirring. Alarge amount of white solid precipitates out which is extracted by ethylacetate. The combined organic layer is further washed by water, brineand dried over sodium sulfate. Solid MeOSuc-Met-Ala-Leu-OBn is obtainedafter removal of solvent.

Example 26

Synthesis of MeOSuc-Met-Ala-Leu

MeOSuc-Met-Ala-Leu-OBn is added into an Erlenmeyer flask with 100 mL ofmethanol. 50 mL of methanol is added. The solution is transferred into ahydrogenation reaction vessel. To this vessel, Pd—C is added. Afterhydrogenation for 2 hours at room temperature, the reaction is stoppedand the catalyst was filtered. Solid MeOSuc-Met-Ala-Leu is yielded afterremoval of solvents.

Example 27

Coupling of MeOSuc-Met-Ala-Leu and Doxorubicin Using the “Urea Method”

Under dry nitrogen atmosphere MeOSuc-Met-Ala-Leu and doxorubicinhydrochloride are suspended/dissolved in 800 mL dry, urea-saturated(˜30% w/v) DMF and DIEA. This mixture is cooled to 0–3° C. over ˜25minutes. At this point HATU is added as a solution in ˜100 mL ureasaturated DMF over 10 minutes (the volume of this solution should bekept minimal). The reaction mixture is stirred for 10 minutes at −2 to2° C. and poured into 4000 mL ice cold brine, containing 2% v/v aceticacid over approximately five minutes with vigorous stirring. The productis filtered off on a medium porosity fritted glass filter, washedgenerously with water and dried under reduced pressure.

Example 28

Synthesis of MeOSuc-Met-Ala-Leu-Dox Therapeutic Agent

In a round bottom flask, MeOSuc-Met-Ala-Leu and doxorubicin aredissolved in anhydrous DMF. After the mixture is stirred for 5 minutes,DIEA followed by HBTU is added into the solution. The mixture is stirredat room temperature for 4 hrs. DMF is removed by a rotary evaporator andthe residue is taken up in 4.0 mL 1:1 methylenechloride:methanol. Tothis solution, 40 mL of ether is slowly added while stirring. Theprecipitate is collected by suction filtration. The solidMeOSuc-Met-Ala-Leu-Dox is washed with ether (2×10 mL) and dried in avacuum desiccator.

Example 29

Removal of Free Doxorubicin from MeOSuc-Met-Ala-Leu-Dox

MeOSuc-Met-Ala-Leu-Dox, DIEA and anhydrous DMF are placed in a 50 mlflask equipped with a magnetic stir bar. When the MeOSuc-Met-Ala-Leu-Doxhas completely dissolved, isocyanate resin (pre-swollen in 5 mL ofdichloromethane for 5 minutes) is added and the resulting solution isstirred for 2 h at room temperature with periodic HPLC monitoring. WhenHPLC traces indicate that the Dox is completely removed, the reactionmixture is filtered through a frit to remove the resin. The resin iswashed with 10 ml DMF and the DMF washes are combined with the filteredreaction mixture. The filtered reaction mixture washes are thenconcentrated on a rotary evaporator equipped with a high vacuum pump anda 30° C. water bath. The residue is suspended in 5 ml of DMF and thesolution is then slowly added into a rapidly stirred anhydrousdiethylether solution. The product is then filtered over a frit, washedwith diethylether, and dried under reduced pressure to giveMeOSuc-Met-Ala-Leu-Dox.

Example 30

Hydrolysis of MeOSuc-Met-Ala-Leu-Dox Via Use of Cross Linked Enzyme

MeOSuc-Met-Ala-Leu-Dox therapeutic agent and 100 mL DMF are placed in a500 mL flask. The suspension is vigorously agitated with a magneticstirrer. When the MeOSuc-Met-Ala-Leu-Dox therapeutic agent hascompletely dissolved, 400 mL deionized water is added and the resultingsolution stirred at 35° C. A slurry of 1 g washed CLEC-PC (AltusBiologics) the immobilized enzyme is rinsed in three aliquots ofdeionized water then resuspended in 10 mL 20% aqueous DMF prior to use.The resulting suspension is stirred at 35° C. with periodic HPLCmonitoring. When all of the MeOSuc-Met-Ala-Leu-Dox therapeutic agent hasbeen consumed, the reaction mixture is filtered through a 0.45 μM nylonmembrane filter to remove the CLEC-PC enzyme. The CLEC-PC cake is washedwith 3×10 mL methanol and the methanol washes are combined with thefiltered reaction mixture. The filtered reaction mixture plus methanolwashes are then concentrated on a rotary evaporator equipped with a highvacuum pump and a 30° C. water bath. The concentrate is then suspendedin 50 mL deionized water at room temperature and rapidly stirred viamechanical stirrer. To this suspension a solution of 77.8 mg sodiumbicarbonate (0.926 mmol, 0.95 eq.) in 100 mL deionized water is addedover 2 minutes. The suspension is stirred at room temperature 20minutes. The reaction mixture is filtered through a 0.45 μM nylonmembrane filter and the Suc-Met-Ala-Leu-Dox is lyophilized.

Example 31

Hydrolysis of MeOSuc-Met-Ala-Leu-Dox Via Use of Soluble Enzyme

MeOSuc-Met-Ala-Leu-Dox therapeutic agent is suspended in 800 mLHPLC-grade water and homogenized for 60 minutes with an Ultraturrax T8homogenizer to yield a finely divided suspension. This suspension isstirred (500 rpm) at 35° C. and adjusted to pH=6.05 with aq. 76 mMNaHCO₃. 1.0 g C. Antarctica “B” lipase (Altus Biologics) is then addedand the reaction mixture stirred at 35° C. for 48 hours. During the 48hr reaction time, pH is maintained between 5.3 and 6.2 by periodicaddition of 76 mM NaHCO₃ and the reaction is periodically monitored byHPLC. After the reaction is nearly complete, the reaction mixture isthen adjusted to pH=7 with aq. 76 mM NaHCO₃ and filtered through a padof Celite 521. The clarified reaction mixture is then acidified to ca.pH 3 with 5 mL glacial acetic acid. The precipitate is isolated byCelite 521 filtration, subsequently rinsing the Celite pad withmethanol. The methanol solution is filtered through a 10–20 μM frittedglass filter and is dried by rotary evaporation. This product isconverted to the sodium salt by dissolution in 70 mL 76 mM NaHCO₃ (0.95eq.) and lyophilized. The product is identical to that of example 24.

Example 32

Immobilized Candida Antarctica “B” Lipase Hydrolysis ofMeOSuc-Met-Ala-Leu-Dox

30.0 g Candida Antarctica “B” lipase (Altus Biologics) is dissolved in300 mL water and dialyzed against 3×41 of 50 mM aq. NaHCO₃ (pH=6.4). 360mL of Pharmacia NHS-Activated Sepharose 4 Fast Flow is placed in acoarse glass fritted funnel and rinsed with 5×450 mL ice-cold 1 mM aq.HCl. The rinsed NHS-Activated Sepharose is then combined with thedialyzed enzyme solution. The resulting suspension is stirred at ambienttemperature (˜22° C.) for 2.0 hours. The Sepharose/enzyme conjugate isthen isolated on a coarse fritted glass filter and then stirred in 1000mL 100 mM aq. TRIS (pH=7.45) for 15 minutes. This suspension is filteredand incubated with another 1000 mL of 100 mM aqueous TRIS buffer(pH=7.45) at 4° C., overnight. In the morning, the immobilized enzyme isfiltered off and after washing with water, is placed into a 2000 mLthree-necked, round-bottomed flask. 43 g MeOSuc-Met-Ala-Leu-Doxtherapeutic agent is added and the solids are suspended in 800 mLdeionized water. The flask is fitted with an overhead stirrer, and apH-stat set to keep the pH of the reaction mixture between 5.9–6.2 bycontrolling a syringe pump. The syringe pump is charged 0.1 M NaHCO₃.Progress of the reaction is followed by HPLC. After the reaction isnearly complete, the immobilized enzyme is filtered off and the liquidphase is lyophilized. The dry solids are then suspended in ˜11 mL dryTHF and filtered off.

Example 33

Synthesis of N-Cap Allyl-Hemisuccinate

This molecule was prepared according the procedure of Casimir, J. R.,et.al. Tet. Lett. 36(19):3409, (1995). 10.07 g (0.1 mol) succinicanhydride and 5.808 g (0.1 mol) allyl-alcohol were refluxed in 100 mLtoluene for 6 hours. The reaction mixture was concentrated under reducedpressure. 15.5 g; 98%. The resulting material was pure enough to use insubsequent reactions. The purity and identity of the semi-solid productwas confirmed by ¹HNMR and ¹³CNMR, by LC/MS.

Example 34

Synthesis of Allyl-Succinyl-Met-Ala-Leu-Dox.

In a round bottom flask N-Cap-Allylhemisuccinyl form of Met-Ala-Leu anddoxorubicin are dissolved in anhydrous DMF. After the mixture is stirredfor 5 minutes, DIEA followed by HATU is added into the solution. Themixture is stirred at room temperature for 2 hours. DMF is removed by arotary evaporator and the residue taken up in 4.0 ml 1:1 DCM: MeOH. Tothis solution, 100 ml of ether is slowly added while stirring. A redprecipitate forms and is collected by suction filtration. The solid iswashed with ether (2×2 ml) and dried in a vacuum desiccator to give theAllyl-Succinyl-Met-Ala-Leu-Dox therapeutic agent.

Example 35

Preparation of Suc-Met-Ala-Leu-Dox from allyl-succinyl-Met-Ala-Leu-Dox

To a stirred solution of allyl-succinyl-Met-Ala-Leu-Dox in 2 mL THF,under nitrogen atmosphere, tetrakis(triphenylphosphine) palladium isadded as a solid. After 10 minutes the precipitate formed during thereaction is filtered off and washed with THF. The solids areSuc-Met-Ala-Leu-Dox.

Example 36

Synthesis of Sodium Salt of Gl-Met-Ala-Leu-Dox

Piperidine (436 μL, 4.413 mmol) was added to a solution of Fmoc form ofMet-Ala-Leu-Dox (95 mg, 0.088 mmol) in DMF (4.5 mL). After stirring for5 minutes at room temperature, the reaction mixture was cooled to −5° C.and glutaric anhydride (624 mg, 5.472 mmol) was quickly added. The coldbath was removed as soon as the color changed and the mixture wasstirred at room temperature for another 10 min. The DMF was removed byrotary evaporation and the residue dissolved in chloroform (2.5 mL).Diethyl ether (14 mL) was added and the resulting precipitate filtered.The filter cake was washed with diethyl ether, air dried and thenresuspended in water (14 mL). The sodium salt was formed by addition of0.025 M NaOH (4 mL, 0.10 mmol) dropwise to the suspension until completedissolution of the solid. This solution was then lyophilized to give thesodium salt of glutaryl N-cap forn of Oligopeptide 38-Dox therapeuticagent in 97% yield with an HPLC purity of 87% by method D.

Example 37

Large Scale Synthesis of Methyl Succinyl-N-Cap form of Leu-Ala-Leu-DoxTherapeutic Agent

120 mmol Doxorubicin.HCl and 199 mmol MeOSuc-Leu-Ala-Leu are dissolvedin anhydrous DMF (10 L) under nitrogen. 76 mL DIEA (434 mmol) is addedto the reaction mixture and the reaction mixture is stirred for 10minutes at room temperature under nitrogen. The reaction mixture is thencooled to 0° C. over 10 minutes. In a separate flask a solution of 864 gHATU (220 mmol) in DMF (500 mL) is prepared. The HATU solution is addedslowly over 20 minutes to the reaction mixture while the reactionmixture is maintained at 0° C. The reaction mixture is stirred at 0° C.for 30 minutes.

A solution of NaCl (7.5 Kg, at least 30% w/v) in water (25 L) isprepared and cooled to 0° C. The reaction mixture is then slowly addedto the cooled brine solution with vigorous stirring over 120 minutes.The color of the solution must remain red, a blue solution indicatesthat the pH needs adjustment immediately to between 5.8–6.0 by addingacetic acid. The temperature is maintained at approximately 5° C. Thered precipitate is filtered off on a medium porosity fritted glassfilter, washed with water and dried under vacuum pressure over P₂O₅ toyield MeOSuc-Leu-Ala-Leu-Dox.

Example 38

Treatment of MeOSuc-Leu-Ala-Leu-Dox with Ps-isocyanate Beads to RemoveTraces of Doxorubicin

146.4 g PS-isocyanate beads (240 mmol; supplied by Argonaut Lab, SanCarlos, Calif.) are dissolved in 1.5 L of anhydrous DMF and allowed toswell for 5–10 minutes at room temperature. The swelled beads arefiltered through a glass-fritted funnel and washed with additional 500mL of anhydrous DMF. 112 mmol MeOSuc-Leu-Ala-Leu-Dox is dissolved in1000 mL of anhydrous DMF and 12 mmol mL DIEA is added followed by theswelled PS-isocyanate beads. The reaction mixture is stirred at roomtemperature and is monitored using HPLC till the amount of doxorubicinpeak is less than 0.1%. Analytical HPLC analyses are performed usingWater 2690 Column: Waters Symmetry Shield C₈ 3.5 μM 4.6×140 mm (cat#WAT094269), solvent: A-80% aqueous 20 mM ammonium formate (pH=4.5) 20%acetonitrile, solvent: B-20% aqueous 20 mM ammonium formate (pH=4.5) 80%acetonitrile. Column temperature: controlled room temperature, sampleTemperature 4° C., Run time: 37.5 minutes, detector: 254 nm, Flow rate:1.0 mL/min, Injection amount 10 μg (0.5 mg/mL×0.02 mL), Mobile Phase Aand B. Gradient: 37.5 minute linear gradient from 100% mobile phase A to100% mobile phase B with a 7.5 minute equilibration delay.

When the doxorubicin peak is less than 0.1%, the reaction mixture isfiltered through a coarse sintered glass funnel to remove the beads. Abrine solution (at least 30% w/v) of 1.1 kg NaCl in 3.5 L water isprepared and cooled to 0° C. The filtered reaction mixture is thenslowly added to the cooled brine solution with vigorous stirring over 45minutes. The color of the solution must remain red, a blue solutionindicates that the pH needs adjustment immediately to between 5.8–6.0 byadding acetic acid. The red precipitate is filtered through a mediumsintered glass funnel, washed with water and dried under vacuum pressureover P₂O₅ to yield MeOSuc-Leu-Ala-Leu-Dox free of any residualdoxorubicin.

MeOSuc-Leu-Ala-Leu-Dox is dissolved in 1 L MeOH and the methanolsolution is then slowly added to 14 L of cooled ethyl ether withvigorous stirring over 60 minutes. The red precipitate is filteredthrough a medium sintered glass funnel, washed with ether (1 L) anddried under vacuum pressure to yield MeOSuc-Leu-Ala-Leu-Dox. The purityis determined by HPLC, as described in Example 44.

Example 39

Enzymatic Hydrolysis of MeOSuc-Leu-Ala-Leu-Dox to YieldSuc-Leu-Ala-Leu-Dox

The CLEC-CAB (Candida Antartica “B” Lipase) enzyme is purchased (fromAltus Biologics., Boston, Mass.) in solution form, where theconcentration of the enzyme is defined by the weight of dry enzyme permilliliter of solution. The crude enzyme suspension is shaken for fewminutes to obtain a homogenous solution. 504 mL (328 mmol) of thishomogenous solution is aliquoted into a flask. 2.5 L of deionized wateris added and the slurry is stirred for 10 minutes using a magneticstirrer. The enzyme solution is filtered using a coarse glass frittedfunnel, without taking the enzyme to dryness. The enzyme is transferredback into a flask. The enzyme is suspended in water and filtered threemore times.

The enzyme cake is resuspended into 550 mL of deionized water andtransferred into a RB flask. To this suspension, MeOSuc-Leu-Ala-Leu-Dox(106 mmol) is added and the reaction mixture is stirred at roomtemperature (25° C). The pH of the reaction mixture is maintainedbetween 5.8 and 6.1 by a pH-stat equipped with a syringe pump chargedwith 1 N NaHCO₃ solution. Progress of the reaction is followed withperiodic HPLC monitoring, as described in Example 44. The reaction iscontinued until the reaction seems to be complete, as determined byHPLC.

To speed up the reaction, additional CLEC enzyme is required when thereaction is complete. Additional CLEC enzyme (homogenous solution) iswashed in a column format as described above. The enzyme cake isresuspended into 1.1 L of deionized water and added to the reactionmixture. The reaction mixture is stirred at room temperature withperiodic HPLC monitoring and the pH is maintained between 5.8 and 6.1.

Once the reaction is complete, the CLEC enzyme is removed from thereaction mixture by filtration through a 0.2 μM filter and rinsed with500 mL of deionized water. The filtrate is then lyophilized to yieldSuc-Leu-Ala-Leu-Dox.Na.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1. A compound comprising: (1) a therapeutic agent capable of entering atarget cell, (2) an oligopeptide of the formula AA³-AA²-AA¹ wherein theoligopeptide is selected from the group consisting of: Leu-Ala-GIy (SEQID NO: 10), and Leu-Tyr-Leu (SEQ ID NO: 13); (3) a stabilizing group;(4) optionally, a linker group not cleavable by TOP, wherein theoligopeptide is directly linked to the stabilizing group at a firstattachment site of the oligopeptide and the oligopeptide is directlylinked to the therapeutic agent or indirectly linked through the linkergroup to the therapeutic agent at a second attachment site of theoligopeptide, wherein the stabilizing group hinders cleavage of theoligopeptide by enzymes present in whole blood, and wherein the compoundis cleavable by TOP.
 2. The compound of claim 1 wherein the stabilizinggroup is a dicarboxylic or higher order carboxylic acid.
 3. The compoundof claim 1 wherein the stabilizing group is selected from the groupconsisting of: succinic acid, adipic acid, glutaric acid, phthalic acid,diglycolic acid, fumaric acid, naphthalene dicarboxylic acid,pyroglutamic acid, acetic acid, 1-naphthylcarboxylic acid,2-naphthylcarboxylic acid, 1,8-naphthyl dicarboxylic acid, aconiticacid, carboxycinnamic acid, triazole dicarboxylic acid, gluconic acid,4-carboxyphenyl boronic acid, polyethylene glycolic acid, butanedisulfonic acid, nipecotic acid, isonipecotic acid, and maleic acid. 4.The compound of claim 1 wherein the stabilizing group is anon-genetically encoded amino acid having four or more carbons.
 5. Thecompound of claim 1 wherein the stabilizing group is one of asparticacid linked to the oligopeptide at the β-carboxy group of the asparticacid or glutamic acid linked to the oligopeptide at the γ-carboxy groupof the glutamic acid.
 6. The compound of claim 1 wherein the stabilizinggroup is negatively charged or neutral.
 7. The compound of claim 1wherein the stabilizing group reduces interaction between the compoundand endothelial cells that line blood vessels when administered to thepatient.
 8. The compound of claim 1 wherein TOP cleaves the linkagebetween AA³ and AA² of the oligopeptide.
 9. The compound of claim 1wherein the compound is cleaved by TOP under an experimental conditionat a test rate of cleavage of 10–80% of a standard rate of cleavage, thestandard rate of cleavage tested on a test standard by TOP under theexperimental condition, the test standard consisting of a conjugate ofSuc-βAla-Leu-Ala-Leu and the therapeutic agent.
 10. The compound ofclaim 9 wherein the test rate of cleavage is 30–65% of the standard rateof cleavage.
 11. The compound of claim 1 wherein the therapeutic agentis selected from the group consisting of the group consisting ofAlkylating Agents, Antiproliferative agents, Tubulin Binding agents,Vinca Alkaloids, Enediynes, Podophyllotoxins or Podophyllotoxinderivatives, the Pteridine family of drugs, Taxanes, Anthracyclines,Dolastatins, Topoiosomerase inhibitors, Maytanisoids and Platinumcoordination complex chemotherapeutic agents, derivatives of theforegoing and analogs of the foregoing.
 12. The compound of claim 1wherein the therapeutic agent is selected from the group consisting ofDoxorubicin, Daunorubicin, Vinblastine, Vincristine, Calicheamicin,Etoposide, Etoposide phosphate, CC-1065, Duocarmycin, KW-2189,Methotrexate, Methopterin, Aminopterin, Dichloromethotrexate, Docetaxel,Paclitaxel, Epithiolone, Combretastatin, Combretastatin A4 Phosphate,Dolastatin 10, Dolastatin 11, Dolastatin 15, Topotecan, Camptothecin,Mitomycin C, Porfiromycin, 5-Fluorouracil, 6-Mercaptopurine,Fludarabine, Tamoxifen, Cytosine arabinoside, Adenosine Arabinoside,Colchicine, Carboplatin, cis-Platin, Mitomycin C, Bleomycin, Melphalan,Chloroquine, Cyclosporin A, Maytansine, and derivatives of theforegoing, and analogs of the foregoing.
 13. The compound of claim 1wherein the target cell is a tumor or inflammatory cell.
 14. Thecompound of claim 1 being a prodrug having an active portion, whereinthe active portion of the prodrug is more capable of entering the targetcell after cleavage by TOP than prior to cleavage by TOP, the activeportion including at least the therapeutic agent.
 15. The compound ofclaim 14 wherein the active portion of the prodrug consists of thetherapeutic agent.
 16. The compound of claim 14 wherein the activeportion of the prodrug includes the therapeutic agent and at least thelinker group.
 17. The compound of claim 14 wherein the active portion ofthe prodrug includes the therapeutic agent and AA¹ of the oligopeptide.18. The compound of claim 17 wherein the active portion of the prodrugfurther comprises AA² of the oligopeptide linked to AA¹.
 19. Thecompound of claim 1 wherein the oligopeptide is directly linked to thetherapeutic agent.
 20. The compound of claim 1 wherein the oligopeptidesequence is indirectly linked to the therapeutic agent at the secondattachment site of the oligopeptide via a linker group, the linker groupselected from the group consisting of amino caproic acid, hydrazidegroup, an ester group, an ether group, and a sulphydryl group.
 21. Thecompound of claim 1 wherein the compound is selected from the groupconsisting of, Suc-Leu-Ala-Gly-Dox, Suc-Leu-Tyr-Leu-Dox.
 22. Thecompound of claim 1 wherein the compound is resistant to cleavage byCD10.
 23. A compound comprising: (1) a therapeutic agent capable ofentering a target cell, (2) an oligopeptide peptide selected from thegroup consisting of: Leu-Ala-GIy (SEQ ID NO: 10), and Leu-Tyr-Leu (SEQID NO: 13); 3) a stabilizing group, the stabilizing group selected from:(a) a dicarboxylic or higher order carboxylic acid, (b) anon-genetically encoded amino acid having four or more carbons, or (c)one of aspartic acid linked to the oligopeptide at the β-carboxy groupof the aspartic acid or glutamic acid linked to the oligopeptide at theγ-carboxy group of the glutamic acid, and (4) optionally, a linker groupnot cleavable by TOP, wherein the oligopeptide is directly linked to thestabilizing group at a first attachment site of the oligopeptide and theoligopeptide is directly linked to the therapeutic agent or indirectlylinked through the linker group to the therapeutic agent at a secondattachment site of the oligopeptide, wherein the stabilizing grouphinders cleavage of the oligopeptide by enzymes present in whole blood,and wherein the compound is cleavable by TOP.
 24. The compound of claim23 wherein the stabilizing group is selected from the group consistingof: succinic acid, adipic acid, glutaric acid, phthalic acid, diglycolicacid, fumaric acid, naphthalene dicarboxylic acid, pyroglutamic acid,acetic acid, 1-naphthylcarboxylic acid, 2-naphthylcarboxylic acid,1,8-naphthyl dicarboxylic acid, aconitic acid, carboxycinnamic acid,triazole dicarboxylic acid, gluconic acid, 4-carboxyphenyl boronic acid,polyethylene glycolic acid, butane disulfonic acid, nipecotic acid,isonipecotic acid, and maleic acid.
 25. The compound of claim 23 whereinthe compound is resistant to cleavage by CD10.
 26. A pharmaceuticalcomposition comprising: (1) a compound comprising: (a) a therapeuticagent capable of entering a target cell, (b) an oligopeptide peptideselected from the group consisting of: Leu-Ala-GIy (SEQ ID NO: 10), andLeu-Tyr-Leu (SEQ ID NO: 13); (c) a stabilizing group, the stabilizinggroup selected from: (d) optionally, a linker group not cleavable byTOP, wherein the oligopeptide is directly linked to the stabilizinggroup at a first attachment site of the oligopeptide and theoligopeptide is directly linked to the therapeutic agent or indirectlylinked through the linker group to the therapeutic agent at a secondattachment site of the oligopeptide, wherein the stabilizing grouphinders cleavage of the oligopeptide by enzymes present in whole blood,and wherein the compound is cleavable by TOP, and (2) a pharmaceuticallyacceptable carrier.